Lactic acid bacteria producing polysaccharide similar to those in human milk and corresponding gene

ABSTRACT

A lactic acid bacterium having a 16S ribosomal RNA characteristic of the genus Streptococcus, cocci morphology, a growth optimum in the range of about 28° C. to about 45° C., having the ability to ferment D-galactose, D-glucose, D-fructose, D-mannose, and N-acetyl (D)-glucosamine, salicin, cellobiose, maltose, lactose, sucrose and raffinose, and imparting a viscosity of greater than 100 mPa.s at a shear rate of about 293 s −1 . The strain often produces an exopolysaccharide comprising a chain of glucose, galactose and N-acetylglucosamine in a proportion of 3:2:1 respectively. The new strain is identified as  Streptococcus macedonicus . Other characteristics include a total protein profile obtained after culture in an MRS medium for 24 h at 28° C., extraction of the total proteins and migration of the proteins on an SDS-PAGE electrophoresis gel, exhibits a degree of Pearson correlation of at least 78 with respect to bacterium CNCM I-1920 or I-1926. The strain and its secreted polysaccharides can be used in preparing dietary compositions. The present invention further relates to a new exopolysaccharide synthesis operon and the genes thereof isolated from the new species and to transformed cells having inserted nucleotides that encode proteins of the EPS operon or at least one gene thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/548,606, filed Apr. 13, 2000, which is acontinuation of the U.S. national phase of International Application No.PCT/EP98/06636, filed Oct. 9, 1998, the content of both of which isexpressly incorporated herein by reference thereto and claim priority toSwiss Patent Application No. 97203245.2 filed Oct. 17, 1997.

FIELD OF THE INVENTION

[0002] The present invention relates to new species of lactic acidbacteria belonging to the genus Streptococcus, identified herein asStreptococcus macedonicus and its use in the production of foodcompositions. The present invention further relates to a newexopolysaccharide synthesis operon isolated from the new speciesStreptococcus macedonicus and transformed microorganisms containing theoperon or genes thereof.

BACKGROUND OF THE INVENTION

[0003] The identification of lactic acid bacteria is essential in thedairy industry, and consists of differentiating distinctivemorphological, physiological and/or genetic characteristics betweenseveral species.

[0004] The distinctive physiological characteristics for a given speciesof lactic acid bacteria may be determined by various tests including,for example, analyzing their capacity to ferment various sugars and themigration profile of total proteins on an SDS-PAGE type electrophoresisgel (Pot et al., Taxonomy of lactic acid bacteria, in Bacteriocins oflactic acid bacteria, Microbiology, Genetics and Applications, L. DeVuyst and E. J. Vandamme ed., Blackie Academic & Professional, London,1994).

[0005] The migration profile of the total proteins of a given species,determined by SDS-PAGE gel electrophoresis, when compared, with the aidof a densitometer, with other profiles obtained from other species,makes it possible to determine the taxonomic relationships between thespecies. Numerical analysis of the various profiles, for example, withthe GelCompar® software, makes it possible to establish the degree ofcorrelation between the species which is a function of variousparameters, in particular of the algorithms used (GelCompar, version4.0, Applied Maths, Kortrijk, Belgium; algorithms: “Pearson ProductMoment Correlation Coefficient, Unweighted Pair Group Method UsingAverage Linkage”).

[0006] To date, comparative analysis of the total protein profile bySDS-PAGE gel electrophoresis has been thoroughly tested as an effectivemeans for distinguishing between homogeneous and distinct groups ofspecies of lactic acid bacteria (Pot et al., Chemical Methods inProkaryotic Systematics, Chapter 14, M. Goodfellow, A. G. O'Donnell,Ed., John Wiley & Sons Ltd, 1994).

[0007] With this SDS-PAGE method, the preceding experiments have thusshown that when a degree of Pearson correlation of more than 78 (on ascale of 100) is obtained between two strains of lactic acid bacteria,it is justifiably possible to deduce therefrom that they belong to thesame species (Kersters et al., Classification and Identification methodsfor lactic bacteria with emphasis on protein gel electrophoresis, inAcid Lactic Bacteria, Actes du Colloque Lactic '91, 33-40, AdriaNormandie, France, 1992; Pot et al., The potential role of a culturecollection for identification and maintenance of lactic acid bacteria,Chapter 15, pp. 81-87, in: The Lactic Acid Bacteria, E. L. Foo, H. G.Griffin, R. Mollby and C. G. Heden, Proceedings of the first lacticcomputer conference, Horizon Scientific Press, Norfolk).

[0008] By way of example, it was recently possible to divide the groupof acidophilic lactic acid bacteria into 6 distinct species by means ofthis technique (Pot et al., J. General Microb., 139, 513-517, 1993).Likewise, this technique was recently used to establish, in combinationwith other techniques, the existence of several new species ofStreptococcus, such as Streptococcus dysgalactiae subsp. equisimilis,Streptococcus hyo lis sp. nov. and Streptococcus thoraltensis sp. nov(Vandamme et al., Int. J. Syst. Bacteriol., 46, 774-781, 1996; Devrieseet al., Int. J. Syst. Bacteriol., 1997, In press).

[0009] The identification of new species of lactic acid bacteria cannothowever be reduced to a purely morphological and/or physiologicalanalysis of the bacteria. To date, the “Deutsche Sammlung VonMikroorganismen und Zellkulturen GmbH” (DSM, Braunschweig, Germany) hasofficially recorded about 48 different species belonging to the genusStreptococcus (see the list below). All these species possess a 16Sribosomal RNA that is typical of the genus Streptococcus, and may bedivided into distinct and homogeneous groups by means of the SDS-PAGEtechnique mentioned above.

[0010] The present invention relates to the identification, by means ofthe techniques presented above, of a new species of lactic acidbacterium belonging to the genus Streptococcus, and to its use in thedairy industry in general.

[0011] As used herein, “biologically pure culture” means a culture freeof deleterious viable contaminating microorganisms.

SUMMARY OF THE INVENTION

[0012] The present invention relates to a new species of lactic acidbacteria belonging to the genus Streptococcus, identified herein asStreptococcus nacedonicus, and its use in the production of foodcompositions.

[0013]Streptococcus macedonicus has a 16S ribosomal RNA characteristicof the genus Streptococcus. Preferably the 16S ribosomal RNAcharacteristic of the new species Streptococcus macedonicus comprises anucleic acid that is SEQ ID NO:1 or a homologue of SEQ ID NO:1 having1-8 nucleotide substitutions, deletions, or additions, or morepreferably only 1-4, and most preferably only 1-2. Other 16S rRNAcharacteristic of Streptococcus can be found at the GenBank database,for example under the accession numbers AF429762-AF429766.

[0014] The new species has cocci morphology and a growth optimum in therange of about 28° C. to about 45° C., and generally has the ability toferment D-galactose, D-glucose, D-fructose, D-mannose, andN-acetyl(D)-glucosamine, salicin, cellobiose, maltose, lactose, sucroseand raffinose, and imparts a viscosity of greater than 100 mPa.s at ashear rate of about 293 s⁻¹ when used to ferment semi-skimmed milk at38° C. at up to a pH 5.2.

[0015] Preferably the strain of Streptococcus macedonicus has 16Sribosomal RNA has a nucleotide sequence that is SEQ ID NO:1.Furthermore, strains of Streptococcus macedonicus advantageously producean exopolysaccharide having a chain of glucose, galactose andN-acetylglucosamine in a proportion of 3:2:1 respectively. Theseexopolysaccharides are useful in the preparation of food compositions,especially diary products. The polysaccharides can also be hydrolyzedand used in hypoallergenic compositions that are desired for use ininfant products and are similar to polysaccharides found in human milk.

[0016] Strains of Streptococcus macedonicus typically have a totalprotein profile obtained after culture of the bacterium in an MRS mediumfor 24 h at 28° C., extraction of the total proteins and migration ofthe proteins on an SDS-PAGE electrophoresis gel, and exhibit a degree ofPearson correlation of at least 78 with respect to the profile obtainedunder identical conditions with the strain of lactic acid bacterium CNCMI-1920 or I-1926.

[0017] The present invention further relates to a new exopolysaccharidesynthesis operon isolated from the new species Streptococcus macedonicusand identified as SEQ ID NO:4 and to the specific genes and peptidesproduced and identified as SEQ ID NOS:5, 6, 8-13, 15, 18-36.

[0018] In one embodiment of the invention, a biologically pure cultureof a lactic acid bacteria strain has a nucleotide sequence which encodespolypeptides identified by SEQ ID NOS: 18, 20, 22-24, 27, 28, 32, and 34(SM-epsA, C, E-G, J, K, O, and Q), wherein the strain produces anexopolysaccharide comprising a chain of glucose, galactose andN-acetylglucosamine in a proportion of 3:2:1 respectively

[0019] Preferably the strain also comprises a nucleotide sequenceencoding polypeptides identified by SEQ ID NOS:21, 25-26, and 33(SM-epsD, H-I, and P) and still more preferably the strain alsocomprises a nucleotide sequence that encodes the polypeptides identifiedby SEQ ID NOS:19 and 29-31 (SM-epsB and L-N). In one embodiment thestrain comprises SEQ ID NO:4.

[0020] The present invention further encompasses the isolated EPS operon(SEQ ID NO:4), genes thereof, and nucleotide sequences that encode thepeptides of the EPS operon and preferably those identified by SEQ IDNO:25 (SM-epsH), SEQ ID NO:26 (SM-epsI), or SEQ ID NO:35 (SM-epsR).

[0021] Another aspect of the invention is use of the isolatednucleotides, or nucleotide sequences that encode peptides of the EPSoperon, to transform a cell. Preferably the transformed cell is amicroorganism that contains the Streptococcus macedonicus EPS operon orat least one of the genes of the operon and produces anexopolysaccharide comprising a chain of glucose, galactose andN-acetylglucosamine in a proportion of 3:2:1 respectively when culturedin milk.

[0022] In a further embodiment, the invention relates to any lactic acidbacterium, whose 16S ribosomal RNA is characteristic of the genusStreptococcus; and whose total protein profile, obtained after migrationof the total proteins on an SDS-PAGE electrophoresis gel, ischaracteristic of that of the strain of lactic acid bacterium CNCMI-1920, but distinct from those of the recognized species belonging tothe genus Streptococcus, namely S. acidominimus, S. agalactiae, S.alactolyticus, S. anginosus, S. bovis, S. canis, S. caprinus, S.constellatus, S. cricetus, S. cristatus, S. difficile, S. downei, S.dysgalactiae ssp. dysgalactiae, S. dysgalactiae ssp. equismilis, S.equi, S. equi ssp. equi, S. equi ssp. zooepidemicus, S. equinus, S.ferus, S. gallolyticus, S. gordonii, S. hyointestinalis, S. hyo lis, S.iniae, S. intermedius, S. intestinalis, S. macacae, S. mitis, S. mutans,S. oralis, S. parasanguinis, S. parauberis, S. phocae, S. pleomorphus,S. pneumoniae, S. porcinus, S. pyogenes, S. ratti, S. salivarius, S.sanguinis, S. shiloi, S. sobrinus, S. suis, S. thermophilus, S.thoraltensis, S. uberis, S. vestibularis, S. viridans.

[0023] A further aspect of the invention is use of a strain of lacticacid bacterium according to the invention for the preparation of adietary composition, in particular an acidified milk or a fromage frais,for example.

[0024] The invention also relates to the use of a polysaccharide,capable of being secreted by a lactic acid bacterium according to theinvention, which consists of a chain of glucose, galactose andN-acetylglucosamine in a respective proportion of 3:2:1, for thepreparation of a dietary or pharmaceutical composition.

[0025] The subject of the invention yet further encompasses a dietary orpharmaceutical composition comprising a strain of lactic acid bacteriumaccording to the invention.

[0026] Finally, the subject of the invention is also a dietary orpharmaceutical composition comprising a polysaccharide consisting of achain of glucose, galactose and N-acetylglucosamine in a respectiveproportion of 3:2:1.

DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1 is a photographic depiction of the migration profiles ofthe total proteins of several strains of the new species, on an SDS-PAGEelectrophoresis gel, in comparison with those obtained withStreptococcus thermophilus strains. The degree of filiation of thestrains is indicated with the aid of the Pearson correlation scale andby means of a tree opposite the protein profiles (the degrees of Pearsoncorrelation of 55 to 100 are represented).

[0028]FIG. 2 is a depiction the graditherm for the strain CNCM I-1920.

[0029]FIG. 3 is an alignment of the S. macedonicus I-1923 epsA PCRamplification product (SEQ ID NO:15) (upper strand) to the S.thermophilus Sfi6 epsA sequence (SEQ ID NO:16) (lower strand). Note the10 base-pair deletion at approximately position 830 in the I-1923sequence.

[0030]FIG. 4 is a diagram of an inverted PCR template and primer pairdesign strategy.

[0031]FIG. 5 shows the strategy for the confirmation of the sequencedDNA used.

[0032]FIG. 6 is a schematic map of the S. macedonicus exopolysaccharidesynthesis operon.

[0033]FIG. 7 shows the ribosome-binding sites for the predicted S.macedonicus eps synthesis genes. The sequences were aligned backwardsfrom the translation initiation codon for each gene and the predictedribosome-binding sites are underlined.

[0034]FIG. 8 shows the DNA sequence and predicted translation productsof the S. macedonicus strain I-1923 exopolysaccharide synthesis operon(SEQ ID NO:4). Probable translation initiation and termination codonsare boxed, while predicted ribosome-binding sites are underlined.

[0035]FIG. 9 is an alignment comparison of the S. pneumoniae serotype33f cap33fM protein (SEQ ID NO:17) (upper sequence) to the predicted S.macedonicus SM-epsP protein (SEQ ID NO:31) (lower sequence). Internaltranslation termination sites are indicated with a large X in red.

[0036]FIG. 10 is a comparison of the I-1923 eps operon DNA sequencessurrounding the IS element to the SC147 eps operon without IS element.

[0037]FIG. 11 is a schematic for the synthesis of the repeatingoligosaccharide unit in S. macedonidus I-1923.

DETAILED DESCRIPTION OF THE INVENTION

[0038] The newly discovered species of the invention is of the genusStreptococcus, referred to herein as Streptococcus macedonicus.Identification of Streptococcus macedonicus is preferably demonstratedby comparing the nucleotide sequence of the 16S ribosomal RNA of thebacteria of the invention, or of their genomic DNA that encodes for the16S ribosomal RNA, with those of other genera and species of lactic acidbacteria known to date. More particularly, it is possible to use themethod disclosed in Example 1 below, or alternatively other methodsknown to a person skilled in the art, for example, as set forth inSchleifer et al., System. Appl. Microb., 18, 461-467, 1995; Ludwig etal., System. Appl. Microb., 15, 487-501, 1992. The nucleotide sequenceSEQ ID NO:1 presented in the sequence listing below is an example of a16S ribosomal RNA sequence that is characteristic of the new species oflactic acid bacteria, and exhibits striking similarities with the 16Sribosomal RNA sequences found in the species of Streptococcus recognizedto date. Preferably the 16S ribosomal RNA characteristic of the newspecies Streptococcus macedonicus comprises a nucleic acid that is SEQID NO:1 or a homologue of SEQ ID NO:1 having 1-8 nucleotidesubstitutions, deletions, or additions, or more preferably only 1-4, andmost preferably only 1-2. Other 16S rRNA characteristic of Streptococcuscan be found at the GenBank database, for example under the accessionnumbers AF429762-AF429766.

[0039] The new species according to the invention, which constitutes adistinct and homogeneous new group, can also be differentiated from theother known species belonging to the,genus Streptococcus by means of thetechnique for identification of the total proteins by SDS-PAGE gelelectrophoresis, described above.

[0040] In particular, this new species may give a total protein profile,obtained after culture of the bacterium in an MRS medium for 24 h at 28°C., extraction of the total proteins and migration of the proteins on anSDS-PAGE electrophoresis gel, which exhibits a degree of Pearsoncorrelation of at least 78 (on a scale of 100) with the profile obtainedunder identical conditions with the strain of lactic acid bacterium CNCMI-1920 or I-1926.

[0041] More particularly, this technique consists of (1) isolating allthe proteins (=total proteins) of a culture of lactic acid bacteriumcultured under defined conditions, (2) separating the proteins byelectrophoresis on an SDS-PAGE gel, (3) analyzing the arrangement of thedifferent protein fractions separated with the aid of a densitometerwhich measures the intensity and the location of each band, (4) andcomparing the protein profile thus obtained with those of several otherspecies of Streptococcus which have been obtained, in parallel orbeforehand, under exactly the same operating conditions.

[0042] The techniques for preparing a total protein profile as describedabove, as well as the numerical analysis of such profiles, are wellknown to a person skilled in the art. However, the results are onlyreliable insofar as each stage of the process is sufficientlystandardized. Faced with this requirement, standardized procedures areregularly made available to the public by their authors such as that ofPot et al., as presented during a “workshop” organized by the EuropeanUnion, at the University of Ghent, in Belgium, on 12 to 16 September1994 (Fingerprinting techniques for classification and identification ofbacteria, SDS-PAGE of whole cell protein).

[0043] The software used in the technique for analyzing the SDS-PAGEelectrophoresis gel is of crucial importance since the degree ofcorrelation between the species depends on the parameters and algorithmsused by this software. Without going into the theoretical details,quantitative comparison of bands measured by a densitometer andnormalized by a computer is preferably made with the Pearson correlationcoefficient. The similarity matrix thus obtained may be organized withthe aid of the UPGMA (unweighted pair group method using averagelinkage) algorithm that not only makes it possible to group together themost similar profiles, but also to construct dendograms (see K.Kerster-s, Numerical methods in the classification and identification ofbacteria by electrophoresis, in Computer-assisted Bacterial Systematics,337-368, M. Goodfellow, A. G. O'Donnell Ed., John Wiley and Sons Ltd,1985).

[0044] Preferably, the strains of the new species exhibit a totalprotein profile having a degree of Pearson correlation of at least 85with respect to one of the strains of bacteria of the new species. Forthe biotypes mentioned below, this degree of Pearson correlation caneven exceed 90, for example.

[0045] By means of the SDS-PAGE electrophoresis gel technique foridentification, the new species according to the invention that belongto the genus Streptococcus may be distinguished from all the species ofStreptococcus recognized to date, namely S. acidominimus, S. agalactiae,S. alactolyticus, S. aoginosus, S. bovis, S. canis, S. caprinus, S.constellatus, S. cricetus, S. cristatus, S. difficile, S. downei, S.dysgalactiae ssp. dysgalactiae, S. dysgalactiae ssp. equisimilis, S.equi, S. equi ssp. equi, S. equi ssp. zooepidemicus, S. equinus, S.ferus, S. gallolyticus, S. gordonii, S. hyointestinalis, S. hyo lis, S.iniae, S. intermedius, S. intestinalis, S. macacae, S. mitis, S. mutans,S. oralis, S. parasanguinis, S. parauberis, S. phocae, S. pleomorphus,S. pneumoniae, S. porcinus, S. pyogenes, S. ratti, S. salivarius, S.sanguinis, S. shiloi, S. sobrinus, S. suis, S. thermophilus, S.thoraltensis, S. uberis, S. vestibularis, and S. viridans.

[0046] The new species according to the invention can also bedistinguished by this technique from the lactic acid bacteria which hadbeen previously classified in error in the genus Streptococcus such asS. adjacens (new classification=Abiotrophia adiacens), S. casseliflavus(=Eliterococcus casseliflavus), S. cecorum (=Enterococcus cecorum), S.cremoris (=Lactococcus lactis subsp. cremoris), S. defectivus(=Abiotrophia defectiva), S. faecalis (=Enterococcus faecalis), S.faecium (=Enterococcus faecium), S. gallinarum (=Enterococcusgallinarum), S. garvieae (=Lactococcus garvieae), S. hansenii(=Ruminococcus hansenii), S. lactis (=Lactococcus lactis subsp. lactis),S. lactis cremoris (=Lactococcus lactis subsp. cremoris), S. lactisdiacetilactis (=Lactococcus lactis subsp. lactis), S. morbillorum(=Gemella morbillorum), S. parvulus (=Atopobium parvulum), S. plantarum(=Lactococcus plantarum), S. raffinolactis (=Lactococcus raffinolactis)and S. saccharolyticus (=Enterococcus saccharolyticus).

[0047] The lactic acid bacteria according to the invention have amorphology characteristic of Lactococcus lactis, for example; that is tosay that they have the shape of cocci assembled into chains.

[0048] The sugars which can be fermented by the new species aregenerally at least one of the following; D-galactose, D-glucose,D-fructose, D-mannose, N-acetyl-(D)-glucosamine, salicin, cellobiose,maltose, lactose, sucrose or raffinose.

[0049] Among all the strains of the new species which have been isolatedin dairies in Switzerland, 7 were deposited under the treaty ofBudapest, by way of example, in the Collection Nationale de Culture deMicroorganisms (CNCM), 25 rue du docteur Roux, 75724 Paris, on 14 Oct.1997, where they were attributed the deposit numbers CNCM I-1920,I-1921, I-922, I-1923, I-1924, I-1925 and I-1926.

[0050] The strains of the new species can be used, for example, toprepare a dietary or pharmaceutical product, in particular in the formof a fresh, concentrated or dried culture.

[0051] Milk-based products are obviously preferred within the frameworkof the invention. Milk is however understood to mean that of animalorigin, such as cow, goat, sheep, buffalo, zebra, horse, donkey, orcamel, and the like. The milk may be in the native state, areconstituted milk, a skimmed milk or a milk supplemented with compoundsnecessary for the growth of the bacteria or for the subsequentprocessing of fermented milk, such as fat, proteins of a yeast extract,peptone and/or a surfactant, for example. The term milk also applies towhat is commonly called vegetable milk, that is to say extracts of plantmaterial which have been treated or otherwise, such as leguminous plants(soya bean, chick pea, lentil and the like) or oilseeds (colza, soyabean, sesame, cotton and the like), which extract contains proteins insolution or in colloidal suspension, which are coagulable by chemicalaction, by acid fermentation and/or by heat. Finally, the word milk alsodenotes mixtures of animal and vegetable milks.

[0052] Pharmaceutical products means products intended to beadministered orally, or even topically, which comprise an acceptablepharmaceutical carrier to which, or onto which, a culture of the newspecies is added in fresh, concentrated or dried form, for example.These pharmaceutical products may be provided in the form of aningestible suspension, a gel, a diffuser, a capsule, a hard gelatincapsule, a syrup, or in any other galenic form known to persons skilledin the art.

[0053] Moreover, some strains of the new species according to theinvention, representing a new biotype of this species, may also have theremarkable property of being both mesophilic and thermophilic(mesophilic/thermophilic biotype). The strains belonging to this biotypehave a growth optimum from about 28° C. to about 45° C. This propertycan be easily observed (1) by preparing several cultures of amesophilic/thermophilic biotype in parallel, at temperatures rangingfrom 20 to 50° C., (2) by measuring the absorbance values for the mediaafter 16 h of culture, for example, and (3) by grouping the results inthe form of a graph representing the absorbance as a function of thetemperature (graditherm). FIG. 2 is particularly representative of thegraphs, which can be obtained with this type of mesophilic/thermophilicbiotype according to the invention. As a guide, among the strains of thenew species having this particular biotype, the strains CNCM I-1920,I-1921 and I-1922 are particularly representative, for example.

[0054] The use of a mesophilic/thermophilic biotype in the dairyindustry is of great importance. Indeed, this species may be used forthe preparation of mesophilic or thermophilic starters. It is thuspossible to produce industrially acidified milks at 45° C. in order toobtain a “yogurt” type product. It is also possible to industriallyproduce cream cheese by fermenting a milk in the presence of rennet at28° C., and separating therefrom the curd thus formed by centrifugationor ultrafiltration. The problems of clogging of the machines linked tothe use of thermophilic ferments are thus eliminated (these problems aredisclosed in patent application EP No. 96203683.6).

[0055] Moreover, other strains of the new species according to theinvention, representing another new biotype of this species, may exhibitthe remarkable property of conferring viscosity to the fermentationmedium (texturing biotype). The viscous character of a milk fermented bya texturing biotype according to the invention may be observed anddetermined as described below:

[0056] 1. Comparison of the structure of a milk acidified by a texturingbiotype with that of milk acidified by non-texturing cultures; thenon-viscous milk adheres to the walls of a glass cup, whereas theviscous milk is self-coherent.

[0057] 2. Another test may be carried out using a pipette. The pipetteis immersed in the acidified milk, which is drawn up in a quantity ofabout 2 ml, and then the pipette is withdrawn from the milk. The viscousmilk forms a rope between the pipette and the liquid surface, whereasthe non-viscous milk does not give rise to this phenomenon. When theliquid is released from the pipette, the non-viscous milk forms distinctdroplets just like water, whereas the viscous milk forms droplets endingwith long strings, which go up to the tip of the pipette.

[0058] 3. When a test tube filled up to roughly a third of a rotaryshaker, the non-viscous milk climbs up the inner surface of the wall,whereas the rise of the viscous milk is about zero.

[0059] The viscous character of this particular biotype may also bedetermined with the aid of a rheological parameter measuring theviscosity. A few commercial apparatus are capable of determining thisparameter, such as the rheometer Bohlin VOR (Bohlin GmbH, Germany). Inaccordance with the manufacturer's instructions, the sample is placedbetween a plate and a truncated cone of the same diameter (30 mm, angleof 5.4°, gap of 0.1 mm), then the sample is subjected to a continuousrotating shear rate gradient which forces it to flow. The sample, byresisting the strain, develops a tangential force called shear stress.This stress, which is proportional to the flow resistance, is measuredby means of a torsion bar. The viscosity of the sample is thendetermined, for a given shear rate, by the ratio between the shearstress (Pa) and the shear rate (s⁻¹) (see also “Le Technoscope deBiofutur”, May 1997).

[0060] The tests of rheological measurement of the texturing characterof this biotype have led to the following definition. A lactic acidbacterium belonging to the texturing biotype according to the inventionis a bacterium which, when it ferments a semi-skimmed milk at 38° C. upto a pH of 5.2, gives to the medium a viscosity which is greater than100 mPa.s at a shear rate of the order of 293 s⁻¹, for example. As aguide, the strains CNCM I-1922, I-1923, I-1924, I-1925 and I-1926 areparticularly representative of this texturing biotype for example.

[0061] This texturing biotype is also of great importance in the dairyindustry because its capacity to give viscosity to a dairy product isexceptionally high when it is compared with those of other species oftexturing lactic acid bacteria, in particular with the strainsLactobacillus helveticus CNCM I-1449, Streptococcus thermophilus CNCMI-1351, Streptococcus thermophilus CNCM I-1879, Streptococcusthermophilus CNCM I-1590, Lactobacillus bulgaricus CNCM I-800 andLeuconostoc mesenteroides ssp. cremoris CNCM 1-1692, which are mentionedrespectively in patent applications EP 699689, EP 638642, EP 97111379.0,EP 750043, EP 367918 and EP 97201628.1.

[0062] It is also possible to note that the production of a viscositymay also take place, for some strains, in a very broad temperature rangethat extends from the mesophilic temperatures (25-30° C.) to thethermophilic temperatures (40-45° C.). This characteristic featurerepresents an obvious technological advantage.

[0063] However, some strains belonging to this new texturing biotypeproduce an exopolysaccharide (EPS) of high molecular weight whose sugarcomposition is similar to that found in the oligosaccharides in humanbreast milk. The EPS in fact consists of a chain of glucose, galactoseand N-acetylglucosamine in a proportion of 3:2:1 respectively (A.Kobata, in the Glycoconjugates, Vol. 1, “Milk glycoproteins andoligosaccharides”, p. 423-440, Ed. 1. Horowitz and W. Pigman, Ac. Press,N.Y., 1977). As a guide, the strains CNCM I-1923, I-1924, I-1925 andI-1926 produce this polysaccharide.

[0064] This exopolysaccharide, in native or hydrolyzed form, could thusadvantageously satisfy a balanced infant diet.

[0065] It is possible to prepare a diet for children and/orbreast-feeding infants comprising a milk which has been acidified withat least one strain of lactic acid bacterium producing an EPS consistingof a chain of glucose, galactose and N-acetylglucosamine in a proportionof 3:2:1, respectively, in particular with the strains CNCM I-1924,I-1925 or I-1926, for example.

[0066] It is also possible to isolate this EPS beforehand from a culturemedium of this biotype, and to use it, in native or hydrolyzed form, asan ingredient in an infant diet, for example.

[0067] The isolation of the EPS generally consists of removing theproteins and the bacteria from the culture medium and in isolating apurified fraction of the EPS. It is also possible to carry out theextraction of the proteins and of the bacteria by precipitation with analcohol or trichloroacetic acid followed by centrifugation, while theEPS can be purified by precipitation in a solvent such as acetonefollowed by centrifugation, for example. If necessary, the EPS may alsobe purified, for example, by means of gel filtration or affinitychromatography.

[0068] In the context of the present invention, the isolation of an EPSalso encompasses all the methods of production of an EPS by fermentationfollowed by concentration of the culture medium by drying orultrafiltration, for example. The concentration may be performed by anymethod known to a person skilled in the art, and in particular byfreeze-drying or spray-drying in a stream of hot air, for example. Tothis effect, the methods described in U.S. Pat. No. 3,985,901, EP 298605and EP 63438 are incorporated by reference into the description of thepresent invention.

[0069] Insofar as the maternal oligosaccharides are small in size, itmay be advantageous to carry out beforehand a partial hydrolysis of theEPS according to the invention. Preferably, the hydrolysis conditionsare chosen so as to obtain oligosaccharides having 3 to 10 units ofsugar, that is to say therefore oligosaccharides having a molecularweight on the order of 600 to 2000 Dalton, for example.

[0070] More particularly, it is possible to hydrolyze the EPS accordingto the invention in a 0.5 N trifluoroacetic acid (TFA) solution for30-90 min at 100° C., and then to evaporate the TFA and to recover theoligosaccharides.

[0071] A preferred infant product comprises hydrolyzed protein materialof whey from which allergens, chosen from a group consisting ofalpha-lactalbumin, beta-lactoglobulin, serum albumin and theimmunoglobulins, have not been removed and in which the hydrolyzedprotein material, including the hydrolyzed allergens, exists in the formof hydrolysis residues having a molecular weight not greater than about10,000 Dalton, such that the hydrolyzed material is substantially freeof allergenic proteins and of allergens of protein origin (ahypoallergenic product in accordance with European Directive 96/4/EC;Fritsche et al., Int. Arch. Aller and Appl. Imm., 93, 289-293, 1990).

[0072] It is possible to mix the EPS according to the invention, innative or partially hydrolyzed form, with this hydrolyzed proteinmaterial of whey, and to then incorporate this mixture, in dried form orotherwise, into numerous food preparations for dietetic use, inparticular into foods for infants. EPS can also be mixed with foodsintended primarily for people suffering from allergies.

[0073] The present invention also relates to the isolated EPS operon(SEQ ID NO:4) and genes thereof, which was isolated from the newspecies, Streptococcus macedonicus. The present invention also relatesto homologues EPS genes, which hybridize with SEQ ID NO:4 or the genesthereof, preferably under highly stringent conditions, e.g., washing in0.1.times.SSC/0.1% SDS at 68.degree. C. (Ausubel F. M. et al., eds.,1989, Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc., and John Wiley & sons, Inc., New York, at p. 2.10.3)and encodes a functionally equivalent gene product; and also any DNAsequence that hybridizes to the complement of the coding sequencesdisclosed herein under less stringent conditions, such as moderatelystringent conditions, e.g., washing in 0.2.times.SSC/0.1% SDS at42.degree. C. (Ausubel et al., 1989, supra), yet which still encodes afunctionally equivalent gene product.

[0074] The invention also encompasses DNA vectors that contain any ofthe coding sequences disclosed herein, and/or their complements (i.e.,antisense); DNA expression vectors that contain any of the codingsequences disclosed herein, and/or their complements (i.e., antisense),operatively associated with a regulatory element that directs theexpression of the coding and/or antisense sequences; and geneticallyengineered host cells that contain any of the coding sequences disclosedherein, and/or their complements (i.e., antisense), operativelyassociated with a regulatory element that directs the expression of thecoding and/or antisense sequences in the host cell. Regulatory elementincludes, but is not limited to, inducible and non-inducible promoters,enhancers, operators and other elements known to those skilled in theart that drive and regulate expression. The invention includes fragmentsof any of the DNA sequences discussed or disclosed herein.

[0075] Standard nucleotide isolation techniques well known to thoseskilled in the art can be used to isolate the nucleotide sequencesdisclosed herein or to synthesize them, such as the techniques used inExample 8, as well as suggested primers that can be used.

[0076] In another embodiment of the invention the isolated EPS operonand a gene thereof are used in the production of transformed cellshaving the EPS operon or a gene thereof. Preferably the transformed cellproduces an exopolysaccharide, when cultured in milk, comprising a chainof glucose, galactose and N-acetylglucosamine in a proportion of 3:2:1,respectively, characteristic of Streptococcus macedonicus. Production ofother exopolysaccharides by the transformed cell are anticipated andencompassed by the present invention however.

[0077] Preferably the transformed cell is a microorganism and morepreferably a microorganism suitable for use in diary food productionsuitable for use at temperatures ranging from 20 to 50° C.Transformation/recombination of a nucleic acid molecule into a cell canbe accomplished by any method by which a nucleic acid molecule can beinserted into the cell. Transformation techniques include, but are notlimited to, transfection, electroporation and microinjection.Preparation and isolation techniques are described by Nelson andHousman, in Gene Transfer (ed. R. Kucherlapati) Plenum Press, 1986.

[0078] Recombinant molecules of the present invention, which can beeither DNA or RNA, can also contain additional regulatory sequences,such as translation regulatory sequences, origins of replication, andother regulatory sequences that are compatible with thetransformed/recombinant cell. One or more recombinant molecules of thepresent invention can be used to produce an encoded product. A preferredmethod is by transfecting a host cell with one or more recombinantmolecules of the present invention to form a transformed/recombinantcell.

[0079] Nucleic acid molecules of the present invention can beoperatively linked to expression vectors containing regulatory sequencessuch as transcription control sequences, translation control sequences,origins of replication, and other regulatory sequences that arecompatible with the transformation cell and that control the expressionof nucleic acid molecules of the present invention. In particular,recombinant molecules of the present invention include transcriptioncontrol sequences. Transcription control sequences are sequences, whichcontrol the initiation, elongation, and termination of transcription.Particularly important transcription control sequences are those thatcontrol transcription initiation, such as promoter, enhancer, operatorand repressor sequences. Suitable transcription control sequencesinclude any transcription control sequence that can function in yeast orbacterial cells. A variety of such transcription control sequences areknown to those skilled in the art.

[0080] It may be appreciated by one skilled in the art that use oftransformation DNA technologies can improve expression of transformednucleic acid molecules by manipulating, for example, the number ofcopies of the nucleic acid molecules within a host cell, the efficiencywith which those nucleic acid molecules are transcribed, the efficiencywith which the resultant transcripts are translated, and the efficiencyof post-translational modifications. Transformation techniques usefulfor increasing the expression of nucleic acid molecules of the presentinvention include, but are not limited to, operatively linking nucleicacid molecules to high-copy number plasmids, integration of the nucleicacid molecules into the host cell chromosome, addition of vectorstability sequences to plasmids, substitutions or modifications oftranscription control signals (e.g., promoters, operators, enhancers),substitutions or modifications of translational control signals,modification of nucleic acid molecules of the present invention tocorrespond to the codon usage of the host cell, deletion of sequencesthat destabilize transcripts, and use of control signals that temporallyseparate recombinant cell growth from recombinant enzyme productionduring fermentation. The activity of an expressed recombinant protein ofthe present invention may be improved by fragmenting, modifying, orderivatizing nucleic acid molecules encoding such a protein.

[0081] Additional identifying characteristics of the new species havenow been identified recently. (See Schlegel, L.; Grimont, F.; Ageron,E.; Grimont, P.; and Bouvet, A. (2003) “Reappraisal of the taxonomy ofthe Streptococcus bovis/Streptococcus equinus complex and relatedspecies: description of Streptococcus gallolyticus subsp. gallolyticussubsp. nov., S. gallolyticus subsp. macedonicus subsp. nov. and S.gallolyticus subsp. pasteurianus subsp. nov. ” Int. J. Syst. Evol.microbial. 53(3), 631-645). These identifying characteristics includeadditional phenotypic characterizations.

[0082] Phenotypic characterization of the new species, Streptococcusmacedonicus typically include the following characteristics:gram-positive cocci, non-motile, and non-sporulating. The catalase testis typically negative. The strains of this species generally showhomogeneous growth in buffered glucose and brain heart infusion brothsand do not produce gas in MRS broth. Typically they are non-haemolyticon sheep-blood agar in an aerobic atmosphere and are tellurite-negative.The strains generally produce leucine aminopeptidase andalanyl-phenylalanyl-proline arylamidase and do notproduce—glucuronidase. Further phenotypic characterizations of the newspecies include production of galactosidase (−GAR test) and usuallynegative for—glucosidase. They usually do not hydrolyze aesculin and donot typically produce acid from glycogen or inulin, or produce tannase.They do not produce acid from melibiose. Production of acid frommethyl-D-glucopyranoside and starch is variable. One type strain of thenew species is ACA-DC 206T (=LAB 617T=ATCC BAA-249T=CCUG 39970T=CIP105683T=JCM 11119T=LMG 18488T=HDP 98362T).

[0083] Quantitative DNA-DNA hybridization relatedness test can bedetermined by labeling the DNA in vitro with [3H]ATP, [3H]TTP, [3H]GTPand [3H]CTP using the Megaprime DNA labelling reaction kit (all fromAmersham). Hybridizations of these labelled DNAs with DNA ofrepresentative strains of the S. macedonicus, preferable CNCM I-1920,I-1921, I-1922, I-1923, I-1924, I-1925 or I-1926, and more preferableCNCM I 1923 or I-1924. Preferably the hybridization complex is carriedout in a liquid medium under stringent conditions consisting of 60 ° C.for 16 h, according to a modification of the S1 nuclease/trichloraceticacid precipitation method (Crosa et al., 1973; Grimont et al., 1980).The temperature at which 50% of the reassociated DNAs were hydrolysed byS1 nuclease (Tm) is determined. The difference between the meltingtemperatures of homoduplexes and heteroduplexes (Tm) is one method ofdetermining DNA divergence between strains with high levels of DNArelatedness (Grimont et al., 1980).

[0084] 16S rDNA sequence determination relatedness can be determined byaligning the sequences using the CLUSTAL multiple-sequence method. Adistance matrix can then computed using a Kimura model for nucleotidesubstitution. Alignment with a selection of the available sequences of16S rDNA characteristic of Streptococcus genus, from GenBank andphylogenetic analysis of the 16S rDNA data can be performed with theMEGALIGN program from the DNAstar package.

[0085] The present invention is described in greater detail by theexamples presented below. It goes without saying however, that theseexamples are given by way of illustration of the subject of theinvention and do not constitute in any manner a limitation thereto. Thepercentages are given by weight unless otherwise stated.

EXAMPLES Example 1

[0086] Identification of a New Species of Streptococcus

[0087] Several strains of lactic acid bacteria isolated from variousdairies in Switzerland were the subject of the following genetic andphysiological identification. The methods used as well as the resultsobtained, which are represented below, show that these strains are partof a new Streptococcus group which is sufficiently distinct andhomogeneous for it to be designated as grouping together a new speciesof lactic acid bacterium. By way of example, some strains belonging tothis new species were deposited under the treaty of Budapest in theCollection Nationale de Culture de Microorganismes (CNCM), 25 rue dudocteur Roux, 75724 Paris, on 14 Oct. 1997, where they received theidentification Nos. CNCM I-1920, I-1921, I-1922, I-1923, I-1924, I-1925and I-1926.

[0088] 1. Morphology of the strains isolated: A morphologycharacteristic of Lactococcus lactis, that is to say a shape of cocciassembled into chains, was observed under a microscope.

[0089] 2. Sugar fermentation profile of the strains isolated: The sugarswhich can be fermented by the isolated strains are generallyD-galactose, D-glucose, D-fructose, D-mannose, N-acetyl-(D)-glucosamine,salicin, cellobiose, maltose, lactose, sucrose and raffinose. Thisfermentation profile was similar to that obtained with the speciesLactococcus lactis.

[0090] 3. 16S ribosomal RNA of the strains isolated: The isolatedstrains were cultured in 40 ml of HJL medium at 37° C. for 24 h, thebacteria were harvested by centrifugation, each bacterial pellet wasresuspended in 2.5 ml of TE buffer (10 mM Tris PH 8, 0.1 mM EDTA)containing 10 mg/ml of lysozyme, and the whole was incubated at 37° C.for 1 h. 100 μl of a solution containing 10 mg/ml of proteinase K, 250μl of a solution containing 500 mM EDTA pH 8.0, and 500 μl of a solutioncontaining 10% SDS was then added. The whole was incubated at 60° C. for1 h so as to ensure complete lysis of the bacteria. After having cooledthe mixtures, 2.5 ml of phenol/chloroform was added, and they werecentrifuged for 10 min in a Heraeus centrifuge so as to separate 2phases. The top phase was removed. The chromosomal DNA present in thebottom phase was precipitated by addition of 2.5 ml of a solutioncontaining 96% ethanol, and the mixture was gently stirred until aprecipitate was formed. The precipitated DNA was removed with the aid ofa wooden toothpick, deposited in a 2 ml Eppendorf tube containing 1 mlof a Tris buffer (10 mM Tris HCl pH 8.0, 10 mM EDTA and 10 μg/ml ofRNase A), and incubated at 56° C. for 1 h. After cooling, the varioussuspensions of DNA were extracted with 1 ml of phenol/chloroform asdescribed above, and the chromosomal DNA was precipitated with ethanol.The DNA was resuspended in an Eppendorf tube containing a quantity of TEbuffer such that the final quantity of DNA for each strain isolated wasabout 250 μg/ml.

[0091] An aliquot of 1 μl of DNA of each strain isolated was amplifiedby PCR with the primers having the respective nucleotide sequences SEQID NO:2 and SEQ ID NO:3 (see sequence listing), for 30 cycles (95° C./30sec, 40° C./30 sec and 72° C./2 min) using Pwo polymerase fromBoehringer. The PCR products were purified with the aid of the QIAGENQIAquick kit, and the products were eluted in 50 μl of TE buffer. Asample of 20 μl of each product was digested with the restrictionenzymes BamHI and SalI, and the 1.6 kb fragments were separated on anagarose gel (1%), and purified with the aid of the QIAGEN QIAquick kit.The fragments were then cloned into the E. coli vector pK19 (R. D.Pridmore, Gene 56, 309-312, 1987) previously digested with BamHI andSalI and dephosphorylated, and competent cells of E. coli strain BZ234(University of Basel collection, Switzerland) were transformed with eachligation product. The transformants were selected for at 37° C. on LBmedium with 50 μg/ml of kanamycin, 30 ng/ml of X-gal and 10 ng/ml ofIPTG. The white colonies containing the insert were cultured for 10 h onLB medium with 50 μl/ml of kanamycin, and the plasmid DNAs were isolatedwith the aid of the QIAGEN QIAprep8 kit.

[0092] A 4 μl sample of each plasmid (1 pmol/μl: obtained from eachstrain isolated) were mixed with 4 μl of labelled primers IRD-41(sequencing primers: MWG Biotech) and 17 μl of H₂O. For each strainisolated, 4 aliquots of 6 μl were added to 4 wells of 200 μl, and 2 μlof a reaction mixture (Amersham; RPN2536) was then added to the wells.The mixtures were amplified by PCR in the Hybaid Omn-E system with 1cycle of 95° C. for 2 min followed by 25 cycles of 95° C./30 sec, 50°C./30 sec and 72° C./1 min. The reaction products were then separatedconventionally on a polyacrylamide gel, and the DNA sequence wasdetermined for each isolated strain. The DNA fragments thus sequencedrepresented the genomic part of the 16S ribosomal RNA.

[0093] The results show that all the strains isolated contain anucleotide sequence similar, or even identical, to the sequenceidentified in SEQ ID NO:1 which is disclosed in the sequence listing.These sequences exhibit numerous homologies with the 16S RNA sequencesfound in the species of lactic acid bacteria belonging to the genusStreptococcus, which leads to these strains being classified in thegenus Streptococcus. For example, Streptococcus thermophilus 95% ID,Lactobacillus Lactis 89% ID, Lactobacillus bulgaricus 88% ID,Lactobacillus Helveticus 84% ID, and Lactobacillus Johnsonii 86% ID.

[0094] 4. Identification by SDS-PAGE electrophoresis gel: The tests werecarried out in accordance with the instructions provided by Pot et al.,presented during a “workshop” organized by the European Union, at theUniversity of Ghent, in Belgium, on 12 to 16 Sep. 1994 (fingerprintingtechniques for classification and identification of bacteria, SDS-PAGEof whole cell protein).

[0095] In short, to cultivate the lactic acid bacteria, 10 ml of MRSmedium (of Man, Rogosa and Sharpe) are inoculated with an MRS precultureof each strain of the new species of lactic acid bacterium, as well asof each reference strain covering as many species of Streptococcus aspossible. The media are incubated for 24 h at 28° C., they are plated ona Petri dish comprising a fresh MRS-agar medium, and the dishes areincubated for 24 h at 28° C.

[0096] To prepare the extract containing the proteins of the bacteria,the MRS-agar medium is covered with a pH 7.3 buffer containing 0.008 Mof Na₂HPO₄.12H₂O, 0.002 M of Na₂HPO₄.2H₂O and 8% NaCl. The bacteria arerecovered by scraping the surface of the gelled medium, the suspensionis filtered through a nylon gauze, it is centrifuged for 10 min at 9000rpm with a GSA rotor, the pellet is recovered and taken up in 1 ml ofthe preceding buffer. The pellet is washed by repeating thecentrifugation-washing procedure, finally about 50 mg of cells arerecovered to which one volume of STB buffer pH 6.8 (per 1000 ml: 0.75 gTris, 5 ml C₂H₆OS, 5 g of glycerol) is added, the cells are broken byultrasound (Labsonic 2000), the cellular debris is centrifuged, and thesupernatent containing the total protein is preserved.

[0097] An SDS-PAGE polyacrylamide gel 1.5 mm thick (Biorad-Protean orHoefer SE600), crosslinked with 12% acrylamide in the case of theseparating gel (12.6 cm in height) and 5% acrylamide in the case of thestacking gel (1.4 cm in height), is then conventionally prepared. Forthat, the polymerization of the two gel parts is carried out inparticular in a thermostated bath at 19° C. for 24 h and 1 hrespectively, so as to reduce the gel imperfections as much as possibleand to maximize the reproducibility of the tests.

[0098] The proteins of each extract are then separated on the SDS-PAGEelectrophoresis gel. For that, 6 mA are applied for each platecontaining 20 lanes until the dye reaches a distance of 9.5 cm from thetop of the separating gel. The proteins are then fixed in the gel, theyare stained, the gel is dried on a cellophane, the gel is digitized bymeans of a densitometer (LKB Ultroscan Laser Densitometer, Sweden)linked to a computer, and the profiles are compared with each other bymeans of the GelCompar® software, version 4.0, Applied Maths, Kortrijk,Belgium. Insofar as the tests were sufficiently standardized, theprofiles of the various species of Streptococcus contained in a givenlibrary were also used during the digital comparison.

[0099] The results then show that all the strains tested belonging tothe new species can be distinguished from all of the following species:S. acidominimus, S. adjacens, S. agalactiae, S. alactolyticus, S.anginosus, S. bovis, S. canis, S. caprinus, S. casseliflavus, S.cecorum, S. constellatus, S. cremoris, S. cricetus, S. cristatus, S.defectivus, S. difficile, S. downei, S. dysgalactiae ssp. dysgalactiae,S. dysgalactiae ssp. equisimilis, S. equi, S. equi ssp. equi, S. equissp. zooepidemicus, S. equinus, S. faecalis, S. faecium, S. ferus, S.gallinarum, S. gallolyticus, S. garvieae, S. gordonii, S. hansenii, S.hyointestinalis, S. hyo lis, S. iniae, S. intermedius, S. intestinalis,S. lactis, S. lactis cremoris, S. lactis diacetilactis, S. macacae, S.mitis, S. morbillorum, S. mutans, S. oralis, S. parasanguinis, S.parauberis, S. parvulus, S. phocae, S. plantarum, S. pleomorphus, S.pnemoniae, S. porcinus, S. pyogenes, S. raffinolactis, S. ratti, S.saccharolyticus, S. salivarius, S. sanguinis, S. shiloi, S. sobrinus, S.suis, S. thermophilus, S. thoraltensis, S. uberis, S. vestibularis andS. viridans.

[0100] All the results show that the degree of Pearson correlationbetween the strains deposited is at least 85. As a guide, FIG. 1 depictsa photograph of one of the electrophoresis gels, the filiation in theform of a tree, as well as the degree of Pearson correlation (indicatedon the top left-hand scale). The strains LAB 1550, LAB 1551 and LAB 1553refer specifically to the strains CNCM I-1921, I-1922 and I-1925. Thestrains LMG15061 and LAB 1607 were not deposited at the CNCM, butobviously form part of this new species.

[0101] In short, all the strains isolated clearly form part of ahomogeneous group, which is distinct from the other species belonging tothe genus Streptococcus.

Example 2

[0102] Mesophilic/Thermophilic Biotype

[0103] Some strains isolated in Example 1 represent a new particularbiotype since they exhibit the remarkable property of being bothmesophilic and thermophilic.

[0104] This property may easily be observed (1) by preparing, inparallel, several cultures of a mesophilic/thermophilic biotype in anM17-lactose medium at temperatures ranging from 20 to 50° C., (2) bymeasuring the absorbance values for the media at 540 nm after 16 h ofculture, and (3) by grouping the results in the form of a graphrepresenting the absorbance as a function of the temperature(graditherm).

[0105]FIG. 2 represents the graditherm obtained with the strain CNCMI-1920. All the other strains isolated belonging to this particularbiotype, in particular the strains CNCM I-1921 and I-1922, also givecomparable graditherms.

Example 3

[0106] Texturing Biotype

[0107] Several strains isolated in Example 1 had the remarkable propertyof being extremely texturing. This property was observed with the aid ofthe rheological parameter of viscosity measured with a Bohlin VORrotational rheometer (Bohlin GmbH, Germany).

[0108] For that, some of the strains isolated were cultured in asemi-skimmed milk at 38° C. with a pH up to about 5.2. In accordancewith the manufacturer's instructions, a sample of each culture mediumwas then placed between a plate and a truncated cone of the samediameter (30 mm, angle of 5.4□, gap of 0.1 mm), then the sample wassubjected to a continuous rotating shear rate gradient which forces itto flow. The viscosity of the sample was then determined at a shear rateof 293⁻¹. The results of the rheology tests carried out with some of thestrains isolated demonstrated that the culture media thus fermented hada viscosity greater than 100 mPa.s, or even a viscosity exceeding 200mPa.s in the case of the strains CNCM I-1922, I-1923, I-1924, I-1925 andI-1926.

[0109] For comparison, viscosities of the order of 54, 94, 104, 158 and165 mPa.s were obtained, under the same operating conditions, with thestrains Lactobacillus helveticus CNCM I-1449, Streptococcus thermophilusCNCM I-1351, Streptococcus thermophilus CNCM I-1879, Streptococcusthermophilus CNCM I-1590, Lactobacillus bulgaricus CNCM I-800 andLeuconostoc mesenteroides ssp. cremoris CNCM I-1692, respectively, whichwere mentioned in patent applications EP 699689, EP 638642, EP97111379.0, EP 750043, EP 367918 and EP 97201628.1, respectively (thestrains CNCM I-800 and I-1692 were reputed to be highly texturingstrains).

Example 4

[0110] New Exopolysaccharide

[0111] Some strains isolated in Example 1, belonging to the texturingbiotype, in particular the strains CNCM I-1923, I-1924, I-1925 andI-1926, produced an EPS of high molecular weight whose sugar compositionwas similar to those found in certain oligosaccharides in human breastmilk. Analysis of the sugars constituting this polysaccharide wascarried out in the following manner.

[0112] The strains of the new species were cultured in 10% reconstitutedskimmed milk, with shaking, for 24 h at 30° C., the pH being maintainedat 5.5 by addition of a 2 N NaOH solution. The bacterial cells and theproteins were removed from the culture medium by means of precipitationin an equal volume of a solution of 25% by weight of trichloroaceticacid, followed by centrifugation (10,000 g, 1 h). The EPSs wereprecipitated by addition of an equivalent volume of acetone, followed bysettling for 20 h at 4° C. The EPSs were recovered by centrifugation,and the pellet was taken up in a 0.1 M NH₄HCO₃ solution pH 7, and thesuspension was dialyzed against water for 24 h. The insoluble materialswere then removed by ultracentrifugation, and the retentate containingthe purified EPS was freeze-dried. The quantity of purified EPS,expressed as mg of glucose equivalent, was on the order of 40 mg perliter of culture.

[0113] The molecular weight of the EPS was determined by means ofgel-filtration chromatography with the aid of a Superose-6 columnconnected to an FPLC system (Pharmacia), as described by Stingele etal., J. Bacteriol., 178, 1680-1690, 1996. The results demonstrated thatall the strains CNCM I-1923, I-1924, I-1925 and I-1926 produce an EPS ofa size greater than 2×10⁶ Da.

[0114] 100 mg glucose equivalent of the purified EPS was hydrolyzed in 4N TFA at 125° C. for 1 h, before being derivatized and analyzed by GLCchromatography according to the method described by Neeser et al. (Anal.Biochem., 142, 58-67, 1984). The results demonstrated that the strainsproduced an EPS consisting of glucose, galactose and N-acetylglucosaminein a mean proportion of 3:2:1, respectively.

Example 5

[0115] Infant Product

[0116] A whey, 18% hydrolyzed with trypsine is prepared according to therecommendations of U.S. Pat. No. 5,039,532. It is traditionallyspray-dried in a stream of hot air, and between 0.1 and 10% of the drypurified EPS described in Example 4 is incorporated into it. Thisproduct can be rapidly reconstituted in water. It is particularlysuitable for a diet for children or breast-feeding infants because ofits hypoallergenic and tolerogenic properties to cow's milk, and becauseit is balanced from a carbohydrate composition point of view.

Example 6

[0117] Infant Product

[0118] The dry purified EPS of Example 4 is hydrolyzed in a 0.5 Ntrifluoroacetic acid (TFA) solution for 30-90 min and at 100° C., theTFA is evaporated, the hydrolyzate is suspended in water and theoligosaccharides having 3 to 10 units of sugar (600 to 2000 Dalton) areseparated by ultrafiltration.

[0119] A whey, 18% hydrolyzed with trypsine is prepared according to therecommendations of U.S. Pat. No. 5,039,532. It is traditionallyspray-dried in a stream of hot air, and between 0.1 and 10% of purifiedoligosaccharides described above is incorporated into it. This productcan be rapidly reconstituted in water. It is particularly suitable for adiet for children or breast-feeding infants because of itshypoallergenic and tolerogenic properties to cow's milk, and because itis balanced from a carbohydrate composition point of view.

Example 7

[0120] Pharmaceutical Product

[0121] A pharmaceutical composition is prepared in the form of a capsulemanufactured based on gelatin and water, and which contains 5 to 50 mgof the purified EPS of Example 4 or the purified oligosaccharides ofExample 6.

[0122] An alternative pharmaceutical product is a pastille consisting ofa culture of the freeze-dried strain CNCM I-1924 are prepared and thencompressed with a suitable binding agent. These pastilles areparticularly recommended for restoring intestinal flora of lactic acidbacteria and for satisfying a balanced diet in terms of essentialcomplex carbohydrates.

Example 8

[0123] Isolation and Analysis of the Streptococcus macedonicusExopolysaccharide Synthesis (EPS) Operon and the Genes thereof

[0124] The Streptococcus macedonicus exopolysaccharide synthesis (EPS)operon was identified, cloned and sequenced as described below.Bioinformatic analysis confirmed the presence of numerous genes relatedto established exopolysaccharide production in both food-grade and somepathogenic Streptococcus species. Based on the derived DNA sequence andthe associated bioinformatic analysis, the EPS operon of the newspecies, S. macedonicus responsible for production of a uniqueexopolysaccharide was identified and isolated as described herein.

[0125] An interesting property of S. macedonicus is its ability toproduce and secrete a polysaccharide with interesting texturingproperties and a sugar composition that indicates a potential use ininfant and medical applications. The exopolysaccharide composition ofglucose, galactose and N-acetylglucosamine in a ratio of 3:2:1 issimilar to the sugar composition of maternal milk and would satisfy awell-balanced diet for infant nutrition. The S. macedonicus strain CNCMI-1923 exopolysaccharide has a branched structure with a repeating threesugar backbone and a three sugar side-chain. The oligosacchariderepeating unit structure has been determined and is shown here:

[0126] Exopolysaccharides are produced by a variety of microorganismswhere they may have diverse functions. In the pathogenic bacteriumStreptococcus pneumoniae, the capsular polysaccharide coats the surfaceof the bacterium and protects it from the environment and host defensemechanisms. The importance of the capsule polysaccharide is seen in S.pneumoniae strains devoid of capsule polysaccharide production which areno longer virulent, while harmless strains producing the capsulepolysaccharides at the surface are able to induce the production ofprotective antibodies by the host. In food-grade lactic acid bacteria,the biological advantage of exopolysaccharide production is less wellunderstood. The present invention provides for use of theseexopolysaccharides as natural texturing agents in certain foods.

[0127] Three mechanisms have so far been elucidated for the secretionand assembly of exopolysaccharides in bacteria. In the first pathway, asdetermined for the O-antigen of Salmonella enterica (Reeves P. (1994)Biosynthesis and assembly of lipopolysaccharide, in Bacterial Cell Wall.Ghyusen J.-M. and Hakenbeck R. (eds). Amsterdam: Elsevier Science, pp281-317), the repeat units are individually synthesized by sequentialtransfer of sugars by the transferases onto a lipid carrier in thecytoplasm. The units are then transferred to the periplasmic face wherepolymerization occurs. In the second pathway, as determined for theO-antigen of Escherichia coli O9, N-actetylglucosamine is transferred toundecaprenol phosphate which then serves as the acceptor molecule forthe addition of the sugars (see Kido N., Torgov V. I., Sugiyama T.,Uchiya K., Sugihara H., Komatsu T., Kato N. and Jann K. (1995).Expression of the O9 polysaccharide of Escherichia coli: sequencing ofthe E. coli O9 rfb gene cluster, characterization of mannosyltransferases, and evidence for an ATP-binding cassette transport system.J. Bacteriol. 177:2178-2187). However, the N-actetylglucosamine isremoved before polymerization and is therefore not a component of thefinal polysaccharide. Secretion and polymerization are similar to thefirst example. In the third pathway, as determined for Salmonellaenterica serovar Borreze (Keenleyside W. J. and Whitfield C. (1996) Anovel pathway for O-polysaccharide biosynthesis in Salmonella entericaserovar Borreze. J. Biol. Chem. 271:28581-28592), initiation is as forthe second pathway, but secretion does not use a transporter. Instead,the processive glycosyltransferase may couple the polymerization of thechain to its transport through a pore-like structure in the membrane.

[0128] DNA fragments were cloned or generated by PCR for sequencedetermination and bioinformatic analysis. A single operon ofapproximately 17 Kb pairs containing the essential genetic elements forthe production and secretion of the polysaccharide was identified.

[0129] Materials and Methods

[0130] 1.1 Strains and Grouth Conditions

[0131] The bacterium Streptococcus macedonicus CNCM I-1923 InstituePasteur Collection (also known as NCC2419 and Sc136 strain) and I-1926from Belgian Culture Collection (NCC1965, Sc147 strain) were provided bythe Nestlé Culture Collection and cultivated in HJL medium at 37° C. Thelaboratory Escherichia coli strain XL1-blue (Stratagene Corp. genotype:recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacI^(q)ZΔM15Tn10]) used for all cloning experiments was cultivated in LB medium at37° C. with vigorous shaking.

[0132] 1.2 Chromosomal DNA Preparation from CNCM I-1923

[0133] Total DNA was prepared from S. macedonicus strain CNCM I-1923 forcloning and sequencing from a 40 ml culture as follows. A 24 hr cultureof CNCM I-1923 in HJL medium grown at 37° C. was centrifuged to recoverthe bacteria. The cell pellet was suspended in 2.5 ml of TE buffer (10mM Tris pH 8.0, 1 mM EDTA) containing 10 mg/ml lysozyme and incubated at37° C. for one h. 100 μl of a 10 mg/ml proteinase K solution, 250 μl of500 mM EDTA pH8.0 and 500 μl 10% SDS were added and the solution gentlymixed and incubated at 60° C. for one h. After cooling, the mixture wasextracted once with 2.5 ml of phenol/chloroform mixture, centrifuged at3,000 rpm to separate the phases and the upper phase removed to a cleantube. The DNA was precipitated by adding 6 ml of 95% ethanol with gentlemixing and transferred to a clean tube with a sterile toothpick. Two mlof a solution of 10 mM Tris-HCl pH8.0, 10 mM EDTA and 10 μg/ml RNase Awas added to the DNA and incubated at 60° C. for one h. After cooling,the solution was extracted once with one ml of phenol/chloroform and thechromosomal DNA again precipitated. This final DNA pellet was suspendedin TE buffer to give a final concentration of approximately 500 μg/ml.

[0134] 1.3 Transformation of E. coli

[0135] A fresh over-night culture of XL1-blue was used to inoculate 100ml of LB medium at 1%. This was incubated at 37° C. with vigorousshaking until an OD600 of 1.0 was reached. At this point, the bacteriawere recovered from the culture by centrifugation at 8,000 rpm for 10min in a GSA rotor and a Sorvall HB3 centrifuge. The culture supernatantwas discarded and the bacteria suspended in 100 ml of sterile water at4° C. The bacteria were recovered by centrifugation and the washrepeated a total of three times. The bacteria were finally suspended in2 ml of sterile 10% glycerol and frozen at −80° C. in convenientaliquots.

[0136] Electro-transformation was performed using a BIO-RAD Gene Pulser®with Pulse Controller, 0.2 cm cuvettes and a single pulse of 2,500 V, 25μFD and 200 Ω. The bacteria were removed in 500 μl of LB medium andincubated at 37° C. with shaking before plating.

[0137] 1.4 Cloning and DNA Sequence Determination of the Eps Operon

[0138] 1.4.1 EspA Gene Cloning

[0139] The DNA sequence of the regulatory gene epsA from S. thermophilusSfi6 (Stingele F., Nesser J.-R. and Mollet B. (1996) Identification andcharacterization of the eps (exopolysaccharide) gene cluster fromStreptococcus thermophilus Sfi6. J. Bacteriol. 178:1680-1690), was usedto design the PCR primer pair 6143 (^(5′)ATGAGTTCGCGTACGAATCG^(3′)) (SEQID NO:7) and 6144 (^(5′)ATACAGATTTTAGAGAAGCC^(3′)) (SEQ ID NO:8). Theamplification reaction contained one μl CNCM I-1923 chromosomal DNA (500ng), 6 μl of 2 mM dNTPs, 2 μl of oligo of each oligonucleotide at 100nM/ml, 10 μl 10×SuperTaq reaction buffer, 80 μl H₂O and 0.3 μl SuperTaqDNA polymerase in a 0.5 ml PCR tube. PCR was performed in a Perkin-ElmerDNA Thermal Cycler with 30 cycles of 95° C. for 30 sec, 50° C. for 30sec, 72° C. for 3 min and finally held at 4° C. The PCR reaction waselectrophoresed on a 1% agarose gel and an amplification product ofapproximately 1.2 kb visualised. This amplicon was cut out of the gel,the DNA eluted using the QIAquick gel extraction kit (QIAgen, Productnumber 28704) and ligated into the vector pGem®-T Easy vector system 1(Promega, Product number A1360). After electro-transformation into E.coli strain XL1-blue, transformants were selected on LB platessupplemented with 100 μg/ml ampicillin, 300 ng/ml X-gal(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, Roche MolecularBiochemicals product number 651 745) and 60 ng/ml IPTG(isopropyl-β-D-thiogalactoside, Roche Molecular Biochemicals productnumber 724 815) at 37° C. White colonies, which have a high probabilityof containing DNA inserts, were grown in small-scale 3 ml cultures andthe plasmid DNA extracted using the QIAprep 8 Miniprep kit (QIAgen,Product number 27144). Samples of extracted plasmid were digested withthe restriction enzyme SacI to identify plasmids containing inserts ofthe expected size. A plasmid was chosen and named pGem-T/epsA.

[0140] 1.4.2 Sequencing of pGem-T/epsA

[0141] The plasmid pGem-T/epsA was sequenced using the IRD800 labeledfluorescent forward (5′CTGCAAGGCGATTAAGTTGGG^(3′)) (SEQ ID NO:9) and thereverse (^(5′)GTTGTGTGGAATTGTGAGCGG^(3′)) (SEQ ID NO:10) primers and theThermo Sequenase fluorescent labeled primer cycle sequencing kit with7-deaza-dGTP (Amersham Pharmacia, RPN2538). The cycle sequencing wasperformed on the HyBaid Omn-E PCR machine with a single incubation at95° C. for 5 min, followed by 25 cycles of 95° C. for 30 sec, 50° C. for72° C. for 2 min and finally held at room temperature. After cyclesequencing, the sequences were electrophoresed and analyzed on the LiCorDNA sequencer. The DNA sequences were exported to the GCG suite ofprograms for analysis.

[0142] 1.4.3 Cloning and Identification of pK19-CNCM I-1923/eps

[0143] Using the above sequence information, a clone bank of CNCM I-1923SplI chromosomal DNA fragments was produced and screened by PCR forlarger clones containing the epsA homologue. Three μg of chromosomal DNAwere digested to completion with the restriction enzyme SplI. A 300 ngsample was ligated into the E. coli vector pK19, previously digestedwith the restriction enzyme Asp718, an enzyme that produces a 4base-pair 5′ overhang that is compatible with that generated by SplI.(See Pridmore R. D. (1987) New and versatile cloning vectors withkanamycin-resistance marker. Gene 56:309-312) The ligation mixture waselectro-transformed into frozen competent XL1-blue, plated onto LBplates supplemented with 50 μg/ml kanamycin, Xgal and IPTG and incubatedat 37° C. for 16 hr. White colonies were tooth-picked into 200 μlvolumes of LB medium supplemented with 50 μg/ml kanamycin in microplatesand incubated at 37° C. to produce mini-cultures. Five microplates ofcultures were produced. 20 μl samples were taken from each of the 12wells in a row and pooled into a single microtube. A one μl sample fromeach pool was screened by PCR with the primer pair 6143 and 6144 usingthe conditions described above. Samples of the PCR reactions werevisualised on a 1.5% agarose gel, a PCR positive pool identified and thePCR detection repeated on the 12 individual wells. The bacteria from thePCR positive well were used to inoculate a culture in LB mediumsupplemented with kanamycin for plasmid isolation and restriction enzymemapping and DNA sequence determination.

[0144] 1.4.4 Inverted PCR

[0145] The inverted PCR technique was used to prepare template DNAfragments flanking the SplI clone for DNA sequence analysis. In thistechnique, chromosomal DNA is digested with frequently cuttingrestriction enzymes and ligated in conditions favoring the formation ofcircular products. These circles were then used as template for the PCRreaction with appropriately designed PCR primer pairs. Three μg of CNCMI-1923 chromosomal DNA was digested to completion with the restrictionenzymes EcoRI, HindIII, NsiI and BclI. These digested DNAs were thenphenol extracted, ethanol precipitated and ligated in a 400 μl volumewith 10 units T4 DNA ligase (Roche Molecular Biochemicals, Productnumber 716 359) at 20° C. for 16 h. The ligations were finally phenolextracted, ethanol precipitated and dissolved in 50 μl TE buffer. One μlof the above inverted PCR template was then used as a template forlong-range PCR using primer pairs designed according to the strategyshown in FIG. 4 and the Expand PCR kit (Roche Molecular Biochemicals,Product number 1 681 842) according to the provided instructions. Thetotal PCR reaction was electrophoresed on a preparative 1% agarose gel,strong PCR products cut out, the DNA eluted and used as a template forDNA sequencing using custom labeled primers.

[0146] 1.4.5 Confirmation of the DNA Sequence

[0147] Due to the possibility of rearrangements of foreign DNA whencloned in high copy-number plasmids (such as pK19) in E. coli and theuse of inverted PCR products as sequencing templates, the integrity ofthe DNA sequence was confirmed by the PCR strategy outlined in FIG. 5.PCR primer pairs were designed to amplify approximately 1200 base-pairfragments directly from the CNCM I-1923 genomic DNA. The primer pairswere also positioned so that the amplified fragments overlapped byapproximately 200 base-pairs and in this way completely covered theregion of interest on both strands. The proof-reading thermostablepolymerase Pwo (Roche Molecular Biochemicals, product number 1 644 947)was used to amplify the fragments from CNCM I-1923 chromosomal DNA, thefragments visualised on a 1.0 % agarose gel and compared to thepredicted sizes.

[0148] The PCR amplicons were purified using the QIAquick PCR cleanupkit (Qiagen, Product number 28104), digested with the restrictionenzymes KpnI and BamHI and the DNA fragments resolved of a preparativeone % agarose gel. The corresponding bands were cut out of the gel andthe DNA eluted using the QIAquick gel extraction kit. These DNAfragments were ligated into the vector pK19, previously digested withthe restriction enzymes BamHI and KpnI and dephosphorylated. Theligation was electro-transformed into competent XL1-blue, 500 μl of LBmedium added and the transformed bacteria incubated at 37° C. for 90min. Aliquots of transformed cells were plated onto LB platessupplemented with kanamycin, X-gal and IPTG and incubated at 37° C. for16 h. Small-scale plasmid preparations were made from a selection ofwhite colonies and subjected to restriction enzyme analysis. Finally,two of these plasmids were sequenced, one with the forward and thesecond with the reverse primer so as to detect potential PCR mutations.

[0149] 1.5 Identification of IS Element Insertion Site

[0150] PCR primer pairs, used to confirm the DNA integrity and sequence,were selected from around the IS element and used to verify thechromosomal DNA environment of other isolates of S. macedonicus from theNestlé Culture Collection. The first primer pair has oneoligonucleotide, 9411 (^(5′)ACAGGTACCTTGTCTGGAAATGCAGAG³′) (SEQ IDNO:11), within the epsD gene 5′ to the IS element and the second, 9412(^(5′)CTCGGATCCAACCGCTCTATCTGCTGC^(3′)) (SEQ ID NO:12), within the ISelement. Similarly, the second primer pair has one oligonucleotide, 9413(^(5′)TCCGGTACCTTTCTCTTGTAGTGACCG^(3′)) (SEQ ID NO:13), within the ISelement and the second, 9414 (^(5 ′)CGTGGATCCCGTGACAAACACTACCTG^(3′))(SEQ ID NO:14), within the epsE gene positioned 3′ to the IS element.One of the strains tested, CNCM I-1926, showed no PCR amplificationproduct with either of the primer pairs 9411+9412 and 9413+9414, butproduced a smaller than predicted amplification product with the primerpair 9411+9414, flanking the IS element. The size of this PCR productcorresponded to the predicted size of this genomic region without the ISelement. This PCR product was cloned, its DNA sequence determined andcompared to that of CNCM I-1923.

[0151] 1.6 Bioinformatic Analysis

[0152] The DNA sequence was compiled using the GelAssemble program fromthe GCG suite of programs (Wisconsin Package Version 10.0, GeneticsComputer Group (GCG), Madison, Wis. USA). The consensus sequence wasexported to the GeneWorks program for prediction of open reading frames,which were compared against the bacterial DNA sequence subset of GenBank(release 110.0) and EMBL (release 58.0) databases using the tFastaprogram in the GCG suite. Finally, each potential protein hit wasextracted and compared to the S. macedonicus protein using the Bestfitprogram from GCG.

[0153] 2 Results and Discussion

[0154] 2.1 Identification of the CNCM I-1923 ExopolysaccharideProduction Operon

[0155] 2.1.1 Identification of an EpsA homologue in CNCM I-1923

[0156] The DNA sequence of the EPS operon of from Sfi6 was used todesign PCR primer pairs to amplify the epsA, epsJ, eps L and epsM genesfrom CNCM I-1923 chromosomal DNA. From these PCR reactions, only theepsA primer pairs produced an amplification product whose approximatesize of 1200 base-pairs corresponds well with that predicted from theSfi6 epsA gene (SEQ ID NO:16). This PCR product was cloned, its DNAsequence determined and compared to that of the Sfi6 epsA gene (SEQ IDNO:16). The result of the bioinformatic analysis is shown in FIG. 3,where a very significant 96.8% DNA sequence identity was revealed,indicating that this is a homologue of the Sfi6 epsA gene (SEQ IDNO:16). This analysis also identifies a ten base-pair deletion atposition 834 within the CNCM I-1923 epsA gene and results in thepremature termination of the protein seven amino acids later.

[0157] While the regulatory genes of many polysaccharide synthesisoperons show a significant level of similarity, the role of this DNAsequence in polysaccharide production in CNCM I-1923 could only beproven by cloning and sequencing adjacent genes. To this end, moretemplate DNA surrounding the epsA gene was cloned or generated byinverted PCR and its DNA sequence determined. Bioinformatic analysisconfirmed the presence of genes involved in EPS production surroundingthe epsA gene.

[0158] The DNA sequence, open reading frame prediction and analysis willbe presented in greater detail later, but initial analysis had revealedthe presence of an IS element, two epsA genes and one complete and onetruncated epsB homologues. The arrangement of the genes was confirmed bythe PCR amplification of short overlapping segments directly from theCNCM I-1923 chromosomal DNA. These amplified fragments were also clonedto confirm the remaining ambiguities present in the DNA sequence toproduce a publication and patent quality sequence.

[0159] 2.1.2 DNA Sequencing of the entire CNCM I-1923 ExopolysaccharideOperon

[0160] The present invention provides a DNA sequence (SEQ ID NO:4) of18,372 base pairs of the S. macedonicus operon for the production ofexocellular polysaccharide. This information was derived from a singlecloned DNA fragment supplemented by inverted PCR products. A map of theoperon with its predicted open reading frames is shown in FIG. 6, whilethe DNA sequence plus protein translation products are shown in FIG. 8.

[0161] 2.2 Analysis of the CNCM I-1923 EPS Operon

[0162] 2.2.1 General Structure

[0163] Of the 21 predicted open reading frames, 15 show clearsimilarities to proteins from previously identified exopolysaccharidesynthesis operons from food or pathogenic bacteria. These results arepresented in Table 1, while Table 2 contains physical information of thepredicted proteins and their predicted function, inferred by similarity.FIG. 7 aligns the proposed initiation codons from the predictedtranslation products and also indicates the most probable ribosomebinding site, the DNA sequence motif to which the bacterial ribosomalcomplex attaches as a pre-requisite to translation of the mRNA intoprotein.

[0164] From the remaining predicted translation products, three liewithin the eps operon. One of these appears to be part of an insertionelement, while the remaining two are of no known function. From theflanking predicted translation products, the gene positioned at thestart of the eps operon is translated in the opposite direction andencodes the start of a probable regulator for an unknown operon. Thesecond flanking predicted gene, positioned at the end of the eps operon,shows no significant similarity to any proteins or genes at present inthe databases.

[0165] Analysis of the DNA sequence with the GCG program Stemloop tofind the presence of probable terminator structures revealed only a suchfew structures with a reasonably strong hybridization energies.

[0166] The overall content of G+C nucleotides in this operon isrelatively low, at approximately 34%. TABLE 1 Listing of the predictedproteins from the S. macedonicus eps operon with the Bestfit scores foridentity and, in brackets, similarity. SM- SM- SM- SM- SM- SM- SM- SM-SM- epsA epsB epsC epsD epsE epsF epsG epsH epsI S. epsA epsB epsC epsD— — — — — therm 61.4 73.3 61.6 65.6 Sfi6 (70.7) (79.0) (68.6) (73.1) S.sali cpsA cpsB cpsC cpsD cpsE End of — — — cps 70.0 72.8 61.6 70.0 41.1seq (1) (75.4) (78.6) (68.1) (74.0) (53.2) S. pneu cpsA cpsB cpsC cpsDcpsE cpsF cpsG — — cps 14 55.0 64.3 55.0 59.0 37.0 81.9 55.7 (2) (63.6)(75.6) (65.3) (70.8) (49.8) (88.6) (65.8) S. pneu cps19f cps19f cps19fcps19f cps19f No sim — — — cps A B C D E 19F 53.8 66.0 53.6 59.0 36.7(3) (62.8) (77.3) (66.2) (72.2) (49.7) S. pneu cap33f cap33f cap33fcap33f cap33f — — — — cps A B C D E 33F 55.1 64.7 53.6 56.6 37.3 (4)(63.3) (76.0) (63.5) (68.4) (50.7) S. alag (incom cpsA cpsB cpsC cpsDcpsE cpsF — — eps plete) 72.9 50.7 60.0 41.0 82.6 55.8 (5) 55.6 (80.8)(62.9) (72.6) (52.0) (87.9) (64.3) (61.9) L. — epsC — epsB — epsE epsF —— lactis 27.8 40.6 37.0 43.0 eps (38.9) (50.0) (50.0) (53.1) (6) St. —capC capA capB — — — — aureus 34.1 29.5 35.6 M eps (47.8) (42.4) (49.0)(7) SM-eps SM-eps SM-eps SM-eps SM-eps SM-eps SM-eps SM-eps J K L M N OP Q S. epsI epsA epsA epsB — — — therm 33.3 95.8 98.5 96.1 Sfi6 (44.2)(97.7) (98.5) (96.1) S. sali — — No sim cpsA cpsB — — — cps 93.8 98.1(94.6) (98.1) S. pneu — cpsJ cpsA cpsA cpsB cpsL — — cps 14 38.1 47.556.3 63.2 34.7 (53.2) (56.8) (62.5) (72.3) (47.4) — — S. pneu — —cps19fA cps19fA cps19tB cps 19F 47.1 54.7 63.9 (56.0) (61.0) (72.9) S.pneu cap33fH cap33fJ — — — cap33fL Cap33f cap33fN cps 33F 29.5 32.5 77.9M 95.3 (40.4) (44.9) (84.7) 73.8 (97.5) (81.3) S. alag cpsH cpsH No simcpsX cpsA — — — cps 36.9 34.9 64.6 70.3 (45.6) (46.9) (69.3) (75.5) L.lactis epsG — No sim No sim No sim epsK — — Eps 30.2 33.6 (42.5) (46.5)St. ?— — — — — — — — aureus M cps (1) Streptococcus salivarius: GeneBankAccession X94980 (2) Streptococcus pneumoniae cps14: GeneBank AccessionX85787 (3) Streptococcus pneumoniae cps19F: GeneBank Accession SPU09239(4) Streptococcus pneumoniae cps33F: GeneBank Accession AJ006986 (5)Streptococcus agalactiae strain COH1: GeneBank Accession AB017355 (6)Lactococcus lactis plasmid pNZ4000: GeneBank Accession LLU93364 (7)Staphalococcus aureus M type 1: GeneBank Accession SAU10927

[0167] (1) Griffin A. M., Morris V. J. and Gasson M. J. (1996) ThecpsABCDE genes involved in polysaccharide production in Streptococcussalivarius ssp. thermophilus strain NCBF 2393. Gene 183:23-27.

[0168] (2) Kolkman M. A., Wakarchuk W., Nuijten P. J. and van der ZeijstB. A. (1997) Capsular polysaccharide synthesis in Streptococcuspneumoniae serotype 14: molecular analysis of the complete cps locus andidentification of genes encoding glycosyltransferases required for thebiosynthesis of the tetrasaccharide subunit. Mol. Microbiol. 26:187-208.

[0169] (3) Morona J. K., Morona R. and Paton J. C. (1997)Characterization of the locus encoding the Streptococcus pneumoniae type19F capsular polysaccharide biosynthesis pathway. Mol. Microbiol.23:751-763.

[0170] (4) Llull D., Lopez R., Garcia E. and Munoz R. Data submitted toGenBank, but not found as published.

[0171] (5) Yamamoto S., Miyake K. and Iijima S. Data submitted toGenBank, but not found as published.

[0172] (6) van Kranenburg R., Marugg J. D., van Swam I. I. Willem N. J.and de Vos W. M. (1997) Molecular characterization of theplasmid-encoding eps gene cluster essential for exopolysaccharidebiosynthesis in Lactococcus lactis. Mol. Microbiol. 24:387-397.

[0173] (7) Lin W. S., Cunneen T. and Lee C. Y. (1994) Sequence analysisand molecular characterization of genes required for the biosynthesis oftype 1 capsular polysaccharide in Staphylococcus aureus. J. Bacteriol.176:7005-7016. TABLE 2 Listing of the S. macedonicus exopolysaccharideoperon protein information and the probable functions. Length MassProtein (aa) (da) Probable function SM-epsA 493 53850 BPS operonregulator. SM-epsB 243 28041 Unknown function in EPS operon. SM-epsC 22924884 EPS export. SM-epsD 213 23312 EPS export. SM-epsE 450 52550Putative glucosyl-1-phosphate transferase. SM-epsF 149 17043 Putativegalactosyltransferase. SM-epsG 161 18598 Putative galactosyltransferase.SM-epsH 245 28286 No similarities. SM-epsI 249 28972 No similarities.SM-epsJ 292 34362 Putative glycosyltransferase. SM-epsK 320 36856Putative N-acetylglucosaminyltransferase. SM-epsL — — Partial, EPSoperon regulator. SM-epsM — — Partial, EPS operon regulator. SM-epsN — —Partial, unknown function in EPS operon. SM-epsO 471 52839 Repeatingunit transporter. SM-epsP — — Transmembrane protein. (Three peptides)SM-epsQ 366 42717 UDP-galactopyranoside mutase.

[0174] 5 2.2.1.1 SM-epsA (SEQ ID NO:18)

[0175] The first gene in the eps operon of S. macedonicus, SM-epsA, ispreceded by a good ribosome-binding site and encodes a predicted proteinof 493 amino acids and a mass of 53.85 kDa. The SM-epsA predictedprotein shows similarities to many predicted regulation proteins fromeps and cps operons. These proteins possess a potential‘helix-turn-helix’ DNA-binding motif in their N-terminal section and aretranscription activators that usually negatively regulate their ownexpression. The present invention provides that the SM-epsA gene is theregulator of the eps operon.

[0176] 2.2.1.2 SM-epsB (SEQ ID NO:19)

[0177] The second gene in the operon, SM-epsB, is preceded by a goodribosome-binding site and encodes a predicted protein of 243 amino acids(28.04 kDa). SM-epsB shows strong similarities to many homologousproteins encoded by eps and cps operons (that occupy the same positionin the operon), but to date, no function has yet been assigned to theprotein.

[0178] 2.2.1.3 SM-epsC (SEQ ID NO:20)

[0179] The third gene in the operon, SM-epsC, is preceded by a goodribosome-binding site and encodes a predicted protein of 229 amino acids(24.88 kDa). The SM-epsC protein shows a strong homology to othereps/cps proteins, most of which occupy a similar third position in theoperon. By sequence similarity, these proteins, together with thefollowing protein, SM-epsD, are involved in the regulation of theexopolysaccharide chain length.

[0180] 2.2.1.4 SM-epsD (SEQ ID NO:21)

[0181] The fourth gene in the operon, SM-epsD, is preceded by a goodribosome-binding site and encodes a predicted protein of 213 amino acids(23.31 kDa). The SM-epsD protein contains a so-called P-loop motifrequired for ATP/GTP binding and could be part of the ABC-transporterapparatus. This is consistent with the role of SM-epsD in chain lengthdetermination and transport of the repeating units. Finally, thebioinformatic analysis shows that the SM-epsD protein is truncated inrelation to the related cps proteins, which could indicate that the ISelement has inserted within the SM-epsD gene, close to thecarboxy-terminus. This will be discussed later in relation to the ISelement.

[0182] 2.2.1.5 SM-epsE (SEQ ID NO:22)

[0183] The sixth gene in the operon, SM-epsE, is preceded by a goodribosome-binding site and encodes a predicted protein of 450 amino acids(52.55 kDa). The SM-epsE protein shows strong similarities(approximately 40% identity) to five glucosyl-1-phosphate transferasesfrom the exopolysaccharide synthesis operons of the genus Streptococcus.

[0184] 2.2.1.6 SM-epsF (SEQ ID NO:23)

[0185] The seventh gene in the operon, SM-epsF, is preceded by a goodribosome-binding site and encodes a predicted protein of 149 amino acids(17.04 kDa). The SM-epsF protein shows strong similarities(approximately 80% protein identity) to the S. pneumoniae serotype 14cpsF and S. alagactiae cpsF proteins. See below for predicted function.

[0186] 2.2.1.7 SM-epsG (SEQ ID NO:24)

[0187] The eighth gene in the operon, SM-epsG, is preceded by a goodribosome-binding site and encodes a predicted protein of 161 amino acids(18.60 kDa). The SM-epsG protein shows three strong sequencesimilarities to the S. pneumoniae serotype 14 cpsG, S. alagactiae cpsFand L. lactis epsF proteins (between 43.0 to 55.0% sequence identity).Experimental results obtained with S. pneumoniae serotype 14 show thatthe 14 cpsF and 14 cpsG proteins associate to form an activegalactosyltransferase. The present invention provides that SM-epsF andSM-epsG together encode a galactosyltransferase.

[0188] 2.2.1.8 SM-epsH (SEQ ID NO:25)

[0189] The ninth gene in the operon, SM-epsH, is preceded by a goodribosome-binding site and encodes a predicted protein of 245 amino acids(28.29 kDa). The SM-epsH protein does not show any significantsimilarities to any translated bacterial DNA sequence in the GenBankdata bank.

[0190] 2.2.1.9 SM-epsI (SEQ ID NO:26)

[0191] The tenth gene in the operon, SM-epsI, is preceded by a weakribosome-binding site and encodes a predicted protein of 249 amino acids(28.97 kDa). The SM-epsI protein does not show any significantsimilarities to any translated bacterial DNA sequence in the GenBank orEMBL data banks. The SM-epsI protein contains a sequence motif requiredfor lipoprotein synthesis. In prokaryotes, these proteins aresynthesized with a precursor signal peptide, which is cleaved by aspecific lipoprotein signal peptidase (signal peptidase II). Thepeptidase recognizes a conserved sequence and cuts upstream of a cysteinresidue to which a glyceride-fatty acid lipid is attached (Hayashi S.and Wu H. C. (1990) Lipoproteins in bacteria. J. Bioenerg. Biomembr.22(3): 451-71). Hence SM-epsI is predicted to be a membrane-associatedprotein.

[0192] 2.2.1.10 SM-epsJ (SEQ ID NO:27)

[0193] The eleventh gene in the operon, SM-epsJ, is preceded by a goodribosome-binding site and encodes a predicted protein of 292 amino acids(34.36 kDa). The SM-epsJ protein shows some extended, but low sequencesimilarity to the amino terminal 200 amino acids of the S. pneumoniaeserotype 33F cap33fH protein. This protein is described as aglycosyltransferase.

[0194] 2.2.1.11 SM-epsK (SEQ ID NO:28)

[0195] The twelfth gene in the operon, SM-epsK, is preceded by a goodribosome-binding site and encodes a predicted protein of 320 amino acids(36.86 kDa). The SM-epsK protein shows a good sequence similarity overthe complete length of the S. agalactiae cpsH protein, described as anN-acetylglucosaminyltransferase.

[0196] 2.2.1.12 SM-epsL (SEQ ID NO:29) and SM-epsM (SEQ ID NO:30)

[0197] The thirteenth and fourteenth genes in the operon, SM-epsL andSM-epsM, encode predicted proteins with a very high level of similarityto the S. thermophilus Sfi6 epsA protein, the predicted regulator of theeps operon. It was this gene that was originally isolated using PCRprimers derived from the S. thermophilus Sfi6 operon, and as can be seenin FIG. 3, contains a ten base-pair deletion in the SM-epsA generelative to the Sfi6 epsA gene which accounts for the frame shift andthe presence of two partial proteins. Taken with the fact the both genesare not preceded by recognizable ribosome-binding sites, it is sure thatthis gene does not produce an active regulator for the S. macedonicuseps operon.

[0198] 2.2.1.13 SM-epsN (SEQ ID NO:31)

[0199] The fifteenth gene the operon, SM-epsN, encodes a predictedprotein of approximately 160 amino acids with a very strong similarity(96% identity) to the first 160 amino acids (out of 243 amino acids) ofthe S. thermophilus Sfi6 epsB protein. The SM-epsN gene translationinitiation codon (GTG) is preceded by a good ribosome-binding site. Itis predicated that this epsB homologue is also no longer active.

[0200] 2.2.1.14 SM-epsO (SEQ ID NO:32)

[0201] The sixteenth gene in the operon, SM-epsO, is preceded by a goodribosome-binding site and encodes a predicted protein of 471 amino acids(52.84 kDa). The SM-epsO protein shows a strong similarity to therepeating unit transporter protein from the S. pneumoniae serotype 33Fcps33 fL protein and is most probably involved in the export of therepeating unit.

[0202] 2.2.1.15 SM-epsP (SEQ ID NO:33)

[0203] The seventeenth gene in the operon, SM-epsP, is preceded by astrong ribosome-binding site and encodes a potential protein with astrong, 73.8% identity to the transmembrane protein cap33fM, of the S.pneumoniae 33F cps operon. The SM-epsP protein also contains the P-loopmotif required for ATP/GTP binding. While this protein would normally beexpected to be involved in the transport of the repeating unit, theSM-epsP gene contains two internal translation termination codons thateffectively truncates the protein at positions 49 and 182. Bioinformaticanalysis reveals that the similarity of the SM-epsP protein to cap33fMis continuous through-out the length, but is broken into three separateparts by the presence of the two stop codons as can be seen in FIG. 7.While this situation has been seen in other eps operons, itssignificance is not yet understood.

[0204] 2.2.1.16 SM-epsQ (SEQ ID NO:34)

[0205] The eighteenth and last gene in the operon, SM-epsQ, is precededby a good ribosome-binding site and encodes a predicted protein of 366amino acids (42.72 kDa). This protein shows a strong similarity to theS. pneumoniae serotype 33F cap33fN protein, a predictedUDP-galactopyranose mutase. This enzyme is involved in sugar conversionin lipopolysaccharide biosynthesis where it catalyses the conversion ofUDP-D-galactopyranose into UDP-D-galacto-1,4-furanose (Nassau P. M.,Martin S. L., Brown R. E., Weston A., Monsey D., McNeil M. R. and DuncanK. (1996) Galactofuranose biosynthesis in Escherichia coli K-12:identification and cloning of UDP-galactopyranose mutase. J. Bacteriol.178:1047-1052).

[0206] 2.2.1.17 Flanking Regions

[0207] The two genes flanking the above described S. macedonicusexopolysaccharide genes show no similarity to any previously describedcps or eps genes. The open-reading frame to the 5′ of the S. macedonicuseps operon is transcribed in the opposite direction to the eps operonand contains a potential ‘helix-turn-helix’ DNA-binding motif in itsN-terminal section and is hence probably a transcription activator ofthe adjacent, unrelated operon. The open-reading frame 3′ to the epsoperon shows no significant similarities to any translated bacterial DNAsequence in the GenBank or EMBL data banks.

[0208] 2.2.2 Repeating Unit Synthesis

[0209] From these gene/protein designations, the present inventionprovides a pathway for the synthesis of the oligosaccharide repeatingunit and associate this with the predicted enzymatic activities. Theaddition of each sugar unit requires a unique sugar transferase. The twounidentified genes, SM-epsH (SEQ ID NO:25) and SM-epsI, (SEQ ID NO:29)most probably encode these missing functions. A prediction of theoligosaccharide repeating unit synthesis pathway has been constructedand is shown in FIG. 11.

[0210] 2.2.3 The IS Element of CNCM I-1923 (SEQ ID NO:35)

[0211] FastA analysis of the predicted open reading frames from the EPSoperon of CNCM I-1923 identified one gene with a very high proteinidentity to the transposase from the S. thermophilus insertion sequenceIS 1191 (Guedon G., Bourgoin F., Pebay M., Roussel Y., Colmin C.,Simonet J. M. and Decaris B. (1995) Characterization and distribution oftwo insertion sequences, IS 1191 and iso-IS981, in Streptococcusthermophilus: does intergeneric transfer of insertion sequences occur inlactic acid bacteria co-cultures?. Mol. Microbiol. 16:(1), 69-78).BestFit pairwise comparison of the translated protein sequences revealeda very high 97.95% identity and a 98.21% similarity over the completelength of the proteins. This high level of similarity between the CNCMI-1923 IS element and IS1191 from S. thermophilus is also seen at theDNA sequence level, with a 99.01% identity over 1313 bp and correspondsexactly to the published size of IS1191. IS1191 and our S. macedonicusIS element has 28 bp imperfect terminal inverted repeats and bothelements potential encode a single protein of 391 amino acids, theprobable transposase. This high sequence identity between the two ISelements is a strong evidence for a recent lateral gene transfer betweenthe two species. The IS1191-like element in S. macedonicus has insertedinto the end of the epsD gene, possibly prematurely terminating thisprotein.

[0212] Screening of the remaining S. macedonicus strains in the NestléCulture Collection identified CNCM I-1926 as lacking this IS1191-likeelement described in strain CNCM I-1923. The DNA sequence of this regionwas determined from CNCM I-1926 and confirms the presence an 8 bp targetduplication upon insertion of the element (again, in agreement withIS1191). Additionally, the insertion of the element has caused thepre-mature termination of the epsD gene, eliminating a predicted 45amino acids from the carboxy-terminus. While this protein is importantfor the exopolysaccharide biosynthesis, its truncation has not adverselyaffected the synthesis in strain CNCM I-1923, which was targeted as thehighest (marginally) exopolysaccharide producer.

[0213] 3 Conclusions

[0214] The present invention provides a DNA sequence and bioinformaticanalysis of the exopolysaccharide production operon from the food-gradelactic acid bacterium S. macedonicus. This bacterium produces a branchedpolysaccharide with a composition close to that of human maternal milkand could be interesting to include in infant formulae or in somemedical applications.

[0215] The S. macedonicus exopolysaccharide operon encodes for proteinswith strongest similarities to both food-grade and pathogenicstreptococci, with only very limited similarity to the operons ofLactobacillus bulgaricus or L. helveticus (data provided by theGlycobiology Group and analysis not reported here). Theexopolysaccharide operon of S. macedonicus strain CNCM I-1923 containsidentified elements for almost all the required functions, includingregulation, transferases for the addition of the sugars, a transport andchain length determination system. The operon shows much evidence oflateral gene transfer from streptococci. The most striking evidence isthe presence of the insertion element which shows an extremely highidentity to IS1191 originally identified in S. thermophilus.Furthermore, a region close to the middle of the operon contains DNAsequences with an unusually high identity to genes from the S.thermophilus Sfi6 exopolysaccharide operon. These sequences correspondto the epsA and epsB genes and are probably rearranged/inactive in S.macedonicus as genes corresponding to the homologous function arepresent and complete at the start of the operon (the usual position).

[0216] The bioinformatic analysis identified four of the sixsugar-transferase genes, while two identified protein coding regionsshowed no known sequence similarities. The present invention providesthat these additional coding regions encode two additional S.macedonicus exopolysaccharide sugar-transferase genes.

1 37 1 1522 DNA Streptococcus macedonicus misc_feature (1460)..(1460) nis a, c, g, or t 1 gtcgacagag ttcgatcctg gctcaggacg aacgctggcggcgtgcctaa tacatgcaag 60 tagaacgctg aagactttag cttgctagag ttggaagagttgcgaacggg tgagtaacgc 120 gtaggtaacc tgcctattag tgggggataa ctattggaaacgatagctaa taccgcataa 180 tagtgtttaa cacatgttag agacttaaaa gatgcaattgcatcactagt agatggacct 240 gcgttgtatt agctagttgg tggggtaacg gcctaccaaggcgacgatac atagccgacc 300 tgagagggtg atcggccaca ctgggactga gacacggcccagactcctac gggaggcagc 360 agtagggaat cttcggcaat gggggcaacc tgaccgagcaacgccgcgtg agtgaagaag 420 gttttcggat cgtaaagctc tgttgtaaga gaagaacgtgtgtgagagtg gaaagttcac 480 acagtgacgg taacttacca gaaagggacg gctaactacgtgccagcagc cgcggtaata 540 cgtaggtccc gagcgttgtc cggatttatt gggcgtaaagcgagcgcagg cggtttaata 600 agtctgaagt taaaggcagt ggcttaacca ttgttcgctttggaaactgt taaacttgag 660 tgcagaaggg gagagtggaa ttccatgtgt agcggtgaaatgcgtagata tatggaggaa 720 caccggtggc gaaagcggct ctctggtctg taactgacgctgaggctcga aagcgtgggg 780 agcaaacagg attagatacc ctggtagtcc acgccgtaaacgatgagtgc taggtgttag 840 gccctttccg gggcttagtg ccgcagctaa cgcattaagcactccgcctg gggagtacga 900 ccgcaaggtt gaaactcaaa ggaattgacg ggggccgcacaagcggtgga gcatgtggtt 960 taattcgaag caacgcgaag aacttaccag gtcttgacatcccgatgcta tttctagaga 1020 tagaaagttt cttcggaaca tcggtgacag gtggtgcatggttgtcgtca gctcgtgtcg 1080 tgagatgttg ggttaagtcc cgcaacgagc gcaacccctattgttagttg ccatcattca 1140 gttgggcact ctagcgagac tgccggtgat aaaccggaggaaggtgggga tgacgtcaaa 1200 tcatcatgcc ccttatgacc tgggctacac acgtgctacaatggttggta caacgagtcg 1260 caagccggtg acggcaagca aatctcttaa agccaatctcagttcggatt gtaggctgca 1320 actcgcctac atgaagtcgg aatcgctagt aatcgcggatcagcacgccg cggtgaatac 1380 gttcccgggc cttgtacaca ccgcccgtca caccacgagagtttgtaaca cccgaagtcg 1440 gtgaggtaac cttttaggan ccagccgcct aaggtgggacagatgattgg ggtgaagtcg 1500 taacaaggta accgtaggat cc 1522 2 34 DNAStreptococcus macedonicus 2 atatccgttt tttcgacaga gttygatyct ggct 34 333 DNA Streptococcus macedonicus 3 atatccggat cctacggyta ccttgttacg act33 4 18373 DNA Streptococcus macedonicus 4 acgccaattt ctgaacggaaattcttaaca tcatcaataa tttcatatgt tcgtgtttcg 60 cgaaggaaaa gctcataacgtgacatatct gttccttcta aaagcgaaac aaaagcattg 120 acaacgaaag catagtgctgtgaagatacg ctaaacagtt ctcggtttgt atttttgctc 180 ttataacgtt cctctaaaagagctgtttgt tctaaaattt ggcgagcata agaaagaaac 240 tcaacgccat ccttagtcaaggtaatgcct tttgggttac gaataaaaat ttcaattccc 300 atttcacgct ccaaatctcgtacagcattt gaaaggcttg gttgagtgat aaaaaagctg 360 cttagcagcc tcgttcatgctccctgtttc tactatttta acgatatagt gtaattgttg 420 tattctcata ggcttagtttagcctaaaaa tgaaattccc gcaagtagac aatatcttct 480 tatgacggga gtgctttaaaaacgaatgtt tacattacaa caacaaaatt acaaaaagat 540 aactaaaacg taacaatttagcgattgatt tacttttctt aaaataaaac gcttattttt 600 ttaaataata ctttaggaagcgcatacagt cgtaaaaatt cagaaaatta caaaattgca 660 aaaaacttac aaaagtgctaaaataggaac gttaatatcc ttataggaat cggagattta 720 aaatgtctaa acattcacgtcatagaagac atcataagag ttcacgttca tactctcgtt 780 ttgatacgaa gacgatagtgaatagtgttt tattagtgtt gtttgctttg ttagcgggga 840 ttgcaactta tctcatgtatgccaataata ttctagcttt tcgtcatctg aatattatct 900 acaccgtttt actagttgctgtcttcctca tatctttggt tttgataatt cggaaaaaag 960 ggaaaatcgt tgtgacggttctcttggtta ttttctcgat tgttgcagct atttcgctat 1020 ttgcctttaa atcattggttgatgtggcta atgatatgaa taaatcagcc tcatattcag 1080 aaattgagat gagtgttgtggtgccagcgg atagctcaat ctcagatgtg acagaattat 1140 caagcgttca agcaccaacaaatgctgatg gtagcaatat cgatactttg ctttctcaaa 1200 ttaagtcaga taaaggtattgatttagcga cagaaacagt agattcttat caagccgctt 1260 atgaaaattt gattaatgggtcaagtcaag caatggtttt gaacagtgct tattcaagct 1320 tgcttgaatt atcatataatgattacgaat caaatttaaa gaccatttat acctataaaa 1380 ttaagaagag tgtttcaagcgaagcaaaat catctgatgc taatgtcttt aacatttata 1440 ttagtggtat tgatacctacggatctattt caaccgtttc acgttcagat gttaatatca 1500 tcttgacagt taatatgaatacacataaga ttttgatgac aacagcacca cgggactcat 1560 atgttcaaat tccagacggaggtgcagatc aatacgataa actgacacac gctggtatct 1620 atggtgttga aacatctgaaaagacactag aaaatcttta tggtattgat attgattatt 1680 acgctcgtat caacttcacatcatttatga atctgattga tgctattggt ggtgtgacag 1740 tttataatga tcaggcatttacaagtctcc atggtaatta taattttgaa gttggaaatg 1800 ttaacttaag ctcaggtgaagaagcacttg cttttgttcg tgaacgctat agtcttaata 1860 atggcgacta cgatcgtggtaataatcaaa tcaaagttat tcaagctatt gttaataaat 1920 taacatcgtt aagttcaatttcaaattact caacaattat ttctaccttg caggattcta 1980 ttcaaaccga tatgtcattagatacaatga tgagccttgc taatgctcag cttgattcag 2040 gtaagaaatt taccattacatcacaagaag taactggtac aggttcaaca ggagaattga 2100 cttcttatgc catgccaactgcaagtcttt atatgattca gttggatgat gctagtgtag 2160 caagtgcatc acaagccattaaagatgtta tggaaggtaa gtagatgatt gatattcatt 2220 ctcatattgt ttttgatgtagatgacggac caactactat tgaagaaagt ttagctttgg 2280 ttggggaaag ttatcgtcagggcgtgcgta cgattgtctc aacgtcacat cgccgcaaag 2340 gaatgtttga aacaccagaagataagattt ttgctaattt tagtcaagtc aaagaagctg 2400 ctgaagccaa atatgaaggcttagaaatct tatatggtgg cgaactctac tatagtagcg 2460 atattctgga aagactggaacaacgccaag ttccaagaat gaacgacaca cgttttgcat 2520 tgattgagtt tagtatgacaacaccatgga aagagattca tacagcactt agcaatgtga 2580 ttatgcttgg aattacaccagttgttgctc atatcgaacg ttataatgcg cttgaattta 2640 atgaagaacg tgttaaagaattgattaaca tggggggtta cacacaaatt aatagctcac 2700 atgttctcaa accaaaattatttggtgata aataccatca attcaaaaaa cgagcacgtt 2760 atttcttgga aaaaaatcttgttcattgtg tcgcaagcga tatgcataac cttggaccaa 2820 gaccgccatt tatggataaagctagggaaa tcgttacaaa agattttgga ccaaataggg 2880 catatgctct tttcgaggaaaatcctcaaa ccttattaga aaataaagat ttataggagt 2940 taatatgaat tcaaatgataatgcaagtat cgagattgat gtactctact tgctaagaaa 3000 actttggagt agaaaatttttcattatttt cattgctcta gttgttggga cagtagcttt 3060 gcttggtagt gttttcttcctcaaacctaa gtacacatca acaactcgta tttatgttgt 3120 gagccgaagt agtgatggcagcttaactaa tcaagatttg caagcaggtt cttatcttgt 3180 taatgactat aaagaagtcattacgtcaaa tgaagttttg tcatctgtca ttagtcaaga 3240 aaatctctca ctttcaacaagtgaattgtc aaatatgatt tctgtaaata ttccaacaga 3300 tacacgtgtt atttcaatctctgttgaaga tacagatgcg aaagaagctt ctgatattgc 3360 taacactatc cgtgaagttgctgcagaaaa aatcaaatct gtaaccaagg tagatgatgt 3420 gacaactttg gaagctgccgaagtcgctag caaaccatca tcaccaaata ttaaacgcaa 3480 tgctgcttta ggtgtacttgttggtggttt cttggctatt gttggtattc ttgtgcttga 3540 agtacttgat gaccgtgttcgtcgtccaga agacgtcgaa gaagtgcttg gtatgacact 3600 tttaggagtt gtaccagatattgataaatt ataaggagaa aaattgtaat gccacagtta 3660 gaattagtga gagctaaagctcaaatggtt aaatctatgg aggaatatta caattctatc 3720 cgtaccaata ttcaatttagtggacgtgat ttaaaagtca ttacgttgac ttcggctcaa 3780 tctggcgaag gaaaatcaacaacgtctgtt aatcttgcaa tttcttttgc gcgtgcaggt 3840 ttccgtacac ttttgattgatgcggataca cgtaactcag tcatgtcagg aacgtttaaa 3900 tctaaggaac gttatcaggggttgacaagt ttcttgtctg gaaatgcaga gttgtcagat 3960 gttatttgtg acacaaatattgataatttg atgattattc ctgctgggca agtcccacca 4020 aaccccacat cattgattcaaaacgataac ttcaaagcga tgattgaaat tattcgtgga 4080 ctttacgact atgttatcattgatacacca ccgcttggct tggttattga tgcagctatc 4140 ttagcgcatt actcagacgctagcttgctt gtagtaaaag cgggggctga taaacgtcgt 4200 acagttacaa aactaaaggaacaattggaa caaagtggtt cagctttcct tggcgttatt 4260 ctgaataaat atgatattcaggtagtgtaa aataagttgt gtaaacacaa aaaggaataa 4320 atccgttata gtagagttgcaaaacattac tagaaagaga tttattccta tgactcagtt 4380 taccacagaa ctacttaacttcctagccca aaagcaagat attgatgaat ttttccgtac 4440 ttctcttgaa acagctatgaatgatctgct tcaagcagag ttatcagcct ttttagggta 4500 tgaaccttac gataaattaggctataattc tgggaatagt cgtaacggaa gctatgcacg 4560 gaaattcgaa accaaatatgggactgttca gttgagtatt cctagagatc gtaatgggaa 4620 ctttagtcca gctttgcttcccgcttatgg acgtcgagat gaccacttgg aagagatggt 4680 tatcaaactc tatcaaaccggtgtaacgac tcgagaaatt agtgatatca tcgagcgaat 4740 gtatggtcat cactatagtcctgccacaat ttctaatatc tcaaaagcaa ctcaggagaa 4800 tgtcgctact tttcatgagcgaagcttaga agccaattac tctgttttat ttcttgacgg 4860 aacctatctt ccattaagacgtggaaccgt tagtaaagaa tgtattcata tcgcacttgg 4920 cattacacca gaaggacagaaggctgttct tggatatgaa atcgccccaa atgaaaacaa 4980 tgcttcttgg tccaccctgttagacaagct tcaaaaccaa ggaatccaac aggtttctct 5040 tgtagtgacc gatggcttcaaggggcttga agagattatc aatcaggctt acccattagc 5100 taaacaacaa cgttgcttaattcatattag tcgaaatcta gctagtaaag tgaaacgagc 5160 agatagagcg gttattctggagcaatttaa aacgatttat cgtgctgaaa atttagaaat 5220 ggcagtgcaa gctttagagaactttatctc cgaatggaaa ccaaagtata ggaaagtcat 5280 ggaaagtctg gagaatacggataatctttt aactttttat cagtttccct accagatttg 5340 gcatagtatt tattcgacaaacctcattga gtctcttaac aaagagatca aacgtcaaac 5400 gaaaaagaag attctttttcctaacgagga ggctctggga cgttatttag ttaccctgtt 5460 tgaagattat aatttcaagcaaagtcaacg cacccataaa gggtttggcc aatgtgctga 5520 cacacttgaa agcttatttgattaacattc ttcaactcta cttgagtgtt tacacataat 5580 tattgacagt atcgatattcacttagataa gtatggttca tatggtagtt acggtgggta 5640 tggtagttat ggcaattacggaaaaagtga agaaaaaaca aaaattggta gaggtaacga 5700 aaaaaatagc tgatacttttaccttagaat agggaacagg gagttacatg tatagcgaag 5760 attcgaaaaa gaaagtttattaccttttgt cggatattat agccttagtg ataagttacc 5820 tcatcttagc acaattttatccttatcatt tttttgatag taaattcttt gcagttgttt 5880 ttgggattct gattgtgattgttagtgttt tgagtgatga atactcttca attaaaaatc 5940 gtggttattt aaaagaattaaaagcatctg tgatttatgg tatgaaagtt ttagttttat 6000 ttacttttgt actgatacttggaaaaattc gttttatcca tgacatttca cagatgtctt 6060 atttcttatt ggggcaaatttttattttag taagcctttt tgtcttcatt ggacgtattt 6120 tagttaagaa tcttttcagaagtcatgcaa cggatattaa acaggtagtg tttgtcacgg 6180 attttacgaa tggtaaggaagtcattaaag agcttagcaa ttccaattac catatcgctg 6240 cttatatcag tcgtcgtgataatcctgata tttcacagcc tatcttaaaa agtactaaag 6300 aaattaggga ttttgtggcaaatcaccaag ttgacgagat atttgttgcc aaaaatcacc 6360 aagatgattt tattgaatttgctcattgct taaaattgtt aggaattcca acgacagtag 6420 ctgttgggaa ttattcggacttctatgttg gaaatagtgt tctaaaaaaa gtaggtgata 6480 cgaccttcat aacgacagcattcaatattg taaaattccg tcagattgct ttaaaacgtc 6540 ttatggatat tgcaatagctttagttggct tagtgattac tggtattgta gccattatta 6600 tcacaccgat aatcaagaaacaatcaccag gacctctaat cttcaaacaa aaacgtgttg 6660 gtaaaaacgg taaagtttttgaaatttaca aatttagaag catgtacacc gatgccgaag 6720 aacgcaaaaa agaattactaacacaaaatg atttggatac tgacttaatg tttaagatgg 6780 atgatgaccc tcgtatcttcccatttggac ataagttacg tgattggtca cttgatgaat 6840 taccacaatt tattaatgtcctaaaaggtg aaatgtctgt tgtgggcaca cgtccaccaa 6900 cgcttgacga atatcatcactatgagttac atcatttcaa acgattgaca accaaaccag 6960 gaattactgg tttatggcaagttagcggtc gtagtgacat taccgacttt gaagaagtcg 7020 tagcacttga tatgaagtatatccaaaact ggagcatcag tgaagatatt aaaattattg 7080 ccaaaacatt tggagtcgtactaaaaagag agggaagtaa gtagagtata ttatgaaagt 7140 ttgtttagta ggttcttctggtggacattt ggcacatttg aatatgctaa aacccttttg 7200 gagtgaacat agccgtttccgggttacatt tgataaagaa gacgcaagaa gtgtgttaag 7260 tgatgaaaaa ttttatccgtgttattttcc gactaacaga aattttaaga atttggtaaa 7320 gaacactttc ttagcacttgaaattttaag aaaagaaaaa cctgacgtta ttatttcatc 7380 aggagcagcg gtagcagttccattttttta tctgggtaaa ctgtttggag cgaaaacggt 7440 ttatatcgaa gtatttgatagaatagataa accgactgtg actggaaagt tggtttatcc 7500 agtgacagat aaatttattgttcagtggga ggagatgaaa actgtctatc ccaaagctat 7560 taatctgggg agtattttttaatgattttt gttacagttg gaactcatga acagcccttt 7620 aataggctta ttaaggaagttgatcgttta aaaaaagaag gtattattac agatgaggtt 7680 tttattcaga caggtttttcaacttatgag cctcaatact gtgactggaa aaatattatt 7740 tcttattctg aaatggaagattacatgaat cgtgcagata ttattatcac gcatggtggt 7800 ccagcgacat tcatgggagcaattgctaaa ggaaaaaaac cgattgttgt tccaagacag 7860 gaaaagtttg gagagcatgtaaatgatcat cagcttgagt ttgctgaaca ggtttctgaa 7920 cgatttggaa gtatcgttgtcgtagaagaa attaatgaat tgcaaaatta ttttaattta 7980 gatttaattg tagatgaaagttccaattcg aacaacctaa gatttaatag tcaattaaaa 8040 caagaaatag aaagtttggttagatgaatg attcctaaaa agattcatta ttgttggttt 8100 ggaggaaatc ctcttcctgacagtgtaaaa aattgtataa attcgtggaa aaaattctgt 8160 ccaaattatg aaataatcgaatggaatgaa tcaaattatg atgtacataa aattccatat 8220 atttctgaag cttataaaaataagaaatat gcttttgtat ctgactatgc taggctagat 8280 atcatatata atgagggcgggttttattta gatactgatg ttgaattgtt aaaagcattg 8340 gacgatttaa cttctgaacactgttatatg ggaatggaac aagtgggtcg tgttaatact 8400 ggattaggtt ttggtgcagaaaaaggacat ctttttataa aagaaaatat gcagcaatat 8460 gaagaagttt cttttaatcttaagctacta gaaacatgtg tggatatcac gacaaattta 8520 ttattatcaa aggggttattagtagaaaat tcatatcaaa aaattagtga tgtgtcaatt 8580 tatccaacag attttttttgtccgtttaat atgcaaacac aagaaatggg aataactaaa 8640 aatacttatt caattcatcattatgattca acttggtatg gtaatggtgt tagtgcaata 8700 attaaaaaga aattattaccattaagagtt aaatctcgta tccttattga taaatattta 8760 ggtgaaggct cttatgctaaaatcaaagct attattaaga aatgatattt ttcaaaggag 8820 gatattttgt taactaatattgaatttttt gatatatata tatttcttgt tactctattt 8880 aaaggattgg gagctgaagcaggtaataaa ttatatgttg tagcattttt tataggatct 8940 attgcgattt gtttaaaaatttcaaaggaa aaattttcat ttaatgaact taaaaaagtt 9000 acttttattt tgataatagggctattagat tttattgttg gcaaaagtac aacgtttttg 9060 tttactgcaa ttgcattaagtggacttaaa aatgttaatg aaaatcgagt tatcaaaatt 9120 gctttttgga ctagattattctcttttcta ttaatggtga gtctaagtaa attgaatatt 9180 attaaagata acttgttccttttttatagg gatggccagt ttgtaggaag gcatacattt 9240 ggttatggac atccgaatcaagcgcagagt gctttaacaa ttttgataat acttgctatt 9300 tatctttata atgagaagtttaatattttc cattatatca ttatgattat tatgaacttc 9360 tatttatata gcttaacatattcgcgtaca ggtttcttga tcggagtatt atgtattgtt 9420 ctgggagtgg ttcaaaaaagtaaaaatgta gaaaaaattt ttgctagagt atttaaaaac 9480 tcatattttt gggctgttttagtgacgcta tttatagggt atttttacac taagattcca 9540 caattaaaaa acttagatgaattattcaca ggtaggttgg cttataacaa cactttatta 9600 aataattata ttccgccacttattgggagt tcaaaataca atgagtatgt taatatcgat 9660 aacggtttta tttctttgatatatcaagga ggtattttag catttttgtg gatttcggct 9720 tgtatcataa aattaatgaatgatttttat atccaaaaaa aatttaggga gttgtttttt 9780 atgagcagct ttatagtttatggaatgaca gaaagttttt ttccaaatat tgctgttaat 9840 atctctctta ttttcattggtaaactgata tttaaaactc gcgaggaagt tatgaatgca 9900 taaagttttt atttttacaccgacatacaa tagagtggaa aatctaaaga aattgtatga 9960 gtcactaagg aagcagacttgtaaagagtt tatttggcta attgtcgatg atggttcaaa 10020 tgatggtact gaattttatattagacagtt acgatctgaa tatatttttg atattgtata 10080 cctaaaaaaa gaaaatggaggcaaacatac tgcgtataat ttagctttag attatatggg 10140 aggagaggga tggcatatggttgtagatag cgatgattgg ttagctagca cagctgttga 10200 atgtattatt aaagatatctcctcacttca agttggtaag cttggagttg tatatccaaa 10260 atatagttta actgaagaattacgatggtt acctgagaaa gtaactgaag ttaatattcc 10320 agacataaaa ttgaaatacgggctttcaat cgagactgca attgttatta aaaatttatt 10380 cattggtcaa ttgagacttccttcatttga gggggagaag tttttgtctg aagaaatttt 10440 ttatattatg ctatcggagtttggaaaatt tcttcctctt aatagaagaa tatatttttt 10500 tgaatatcta gaacatggtctaactaataa tctttttcat ctgtggaaga agaaccccaa 10560 gagcacttat ttattgtttaaagagagaaa aaaatatatc ctgcaaaatt tatcaggttt 10620 taaccgaatt gttgaattgtttaaagtgtc cttgaatgaa caagcattat cgctagcaac 10680 atcaaagaat gaaaatattccccaagagct atctgttggg gaacgtatgc taaaaccatt 10740 ggcatattta ttttatttaaaaaggtataa ataggaataa gtatcatagt gaggagatat 10800 tgtggataat gagttaatcagtattattgt tcctgtatac aatgttgaaa aatacattgc 10860 taagtgtttg gactctttagttaaccaaac atatttaaat atagaaatac ttctaattga 10920 tgatggatct acagacaaatcattatcgat atgtaagaag tatgctgcag ttgattctcg 10980 aattaagctt ttttctaaagagaatggcgg cgtttctagc gctcgaaatc taggtcttct 11040 acatgttcaa ggagagtacgttgtgtttgt agattcagat gactttgtat caccaaaata 11100 ttgtgaacat ttatatcaacttactataag tactaagtca gagttagctt ctgtaagtcg 11160 ttataacatt ttgaataaagaggtggtaaa gatatcggat ttatctttta atcaaataac 11220 atcagatgaa gccttaagaaaattcttttt aggtgagggg ataaattgtt atcttttttc 11280 aaaaatattt aaatatgaaactataaaagg actccgattt gatgaaagtt tagaatcagc 11340 agaggacgtt ttgtttatttatcaaactct taagaacata aattttgcat ctatggatgg 11400 cactgttgca gattatttttatattcttag agaaggatct ttaacaaata aaagactgac 11460 ttcatcaaga attgatagttccattagagt tgcggaattt attactagag attgcaacag 11520 caacaaaaaa ttgaaaatgttaagtgaaat taatgaaata tcattaaagg gtgaggttct 11580 tgagtggatt tcattaaatagtgaacttag aattgagttt gaagaatatt ataatatcat 11640 actgagagaa gttagaaagtttaaattgtt acataaagtt caatatctaa ctttaaaaaa 11700 atttattagg attatattattaaaagttag tcctagatta gttacaatct taaaaaataa 11760 ataggtatcc tggaaggagtattcatggat tttaatagta accctcttgt ttcaattatt 11820 attccaattt ataatgtagaaaattattta gaacagtgct ctacttgagt gtttacacat 11880 aattattgac agtatctcacaatataatgg aaaatgatat aaattaaatg attgatatca 11940 taataaaaac tttttcttatgttttgaaaa aagaatgaca attgaaatga agttgtatta 12000 atgttatatt aataataatgggggatatct aattttaatt tttaggagca atttatatga 12060 gttcgcgtac gaatcgtaagcaaaagcata cgagtaatgg atcgtggggg gatggtcaac 12120 gttgggttga ccattctgtatgctatttta gcattggtct tattattcac catgttcaat 12180 tataatttcc tatcctttaggtttttgaac atcattatca ccattggttt gttggtagtt 12240 cttgctatta gcatcttccttcagaagact aagaaatcac cactagtgac aacggttgta 12300 ctggttatct tctcgctagtttctctggtt ggtatttttg gttttaaaca aatgattgat 12360 atcactaacc gtataaatcagactgcagcc ttttcagaag tagaaatgag cattgtggtt 12420 ccgaaggata gtgacatcagagatgtgagt cagattacta gcgttcaggc accaactaag 12480 gttgataaga ataatatcgatagtttgatg tcagctctaa aggaagacaa aaaagttgat 12540 gacaaagttg atgatgtcgcttcctatcaa gaagcctatg acaatcttaa gtctggcaaa 12600 tctaaagcta tggtcttgagtggctcttat gctaccctat tagagtctgt cgatagtaat 12660 tatgcttcaa atctaaaaacaatttatact tataaaatta aaaagaaaaa tagcaactct 12720 gcaaaccaag tagattcaaaagtcttcaat atttatatta gtggtattga tacctacggt 12780 ccgatttcaa cagtatcacgttcagatgtc aatatcatta tgacagtaaa catgaataca 12840 cataagattc tcttgacgactactccacgt gatgcatacg ttaagattgg gcagaccagt 12900 atgataaatt aacccacgcaggtatttatg gcgttgaaac atctgaacaa actctggaag 12960 atctttatgg tattaagattgattactatg cacgaattaa cttcacatct ttccttaagt 13020 tgattgacca acttggtggtgtgacagtcc ataatgatca agctttcaca caagggaagt 13080 ttgatttccc ggttggagatatccaaatga attcagagca agcacttgga tttgttcgtg 13140 aacgctataa tttagatggcggagataatg accgtggtaa aaaccaggag aaagttattt 13200 ctgcgatttt aaacaagttggcttctctaa aatctgtatc aaactttact tcaatcgtta 13260 ataatctcca agactctgttcaaacgaata tgtctttgaa tcccattaac gctttggcta 13320 atacacaact tgaatcaggttctaaattta cggtgacttc tcaagcagta acaggtacag 13380 gttcaaccgg acaattgacctcttatgcga tgccaaattc tagtctttac atgatgaaac 13440 tagataattc gagtgtggaaagtgcctctc aagctatcaa aaaattaatg gaggaaaaat 13500 aagtgattga cgttcactcacatatcgttt ttgatgttga tgatggtcct aaaactttag 13560 aagaaagttt agacctcattggtgaaagtt acgcccaggg ggtacgtaag attgtttcaa 13620 catcccatcg tcgtaagggaatgtttgaga ctccagagaa taaaattttt gccaactttt 13680 ctaaggtaaa agcagaagcagaagcacttt atccagactt aactatttat tatggaggtg 13740 aacttgatta taccttggacattgtggaga aacttgaaaa gaatctcatt ccgcgcatgc 13800 acaacactca atttgctttgattgagttta gtgctcgcac atcttggaaa gaaattcata 13860 gtgggcttag taatgttttgagagcggggg taacacctat tgttgctcat attgagcgct 13920 atgatgccct cgaagaaaatgctgaccgtg ttcgagaaat catcaattac gacactagga 13980 attgcaagta aaaatgggagagtagaatga aagttttaaa aaattacgcc tacaatcttt 14040 cctatcaatt actggtcattgttttaccaa tcattacgac accttatgtt actaggattt 14100 ttagttcaaa ggatttaggtacttatggtt actttaattc gattgtggcc tactttattc 14160 ttttggcaac tttaggtgttgctaactatg gtactaaaga gatttcagga catcgaaagg 14220 atattcgtaa aaatttctggggtatttata ccctccaatt gattgcgact attttgtctc 14280 ttgtcttgta tacatcattatgtttattct ttcctggtat gcaaaatatg gtggcttata 14340 tcttaggatt aagcttgatatcgaaaggaa tggatatttc ttggttattc caaggtttgg 14400 aggattttcg tcgtattaccgcaaggaata caacggtaaa ggttttagga gttatttcta 14460 tcttcctatt tgtgaaaacacctggtgatt tgtatctcta tgttttccta ttgaccttct 14520 ttgaattgct tgggcaattaagtatgtggt taccagcgag accttacatt ggaaaaccac 14580 aatttgattt atcctatgctaagaaacgtc ttaaacctgt tattttgctg tttctccctc 14640 aggttgccat ttcactatacgtgactttgg atcgtacaat gttgggtgcc ttgtcatcga 14700 caaatgatgt agggatttatgatcaggctt tgaaaataat taatattttg ttgacgttgg 14760 tgacttcatt gggaagtgtaatgcttccaa gggtatctgg tcttttatct aacggagatc 14820 ataaggccgt taacaagatgcatgagttgt ctttcttgat ttataatctt gtgatcttcc 14880 cgataatagc aggtctcttgattgttaata aggattttgt gagtttcttc ctagggaaag 14940 atttccaaga ggcttatcttgccattgcta ttatggtctt taggatgttc tttatcggtt 15000 ggacaaatat tatgggaatccagattttga ttccacacaa taaacatcgt gagtttatgc 15060 tctctacgac tattccggctgttgtcagtg ttggacttaa tctcttgtta attcctccat 15120 ttggcttcgt tggtgcctcaattgtatcag ttttaacaga ggctttggta tggttcattc 15180 aattgtactt ctgccttccttacctcaagg aagtaccgat tcttgagtct ttggccaaaa 15240 ttgtatgcgc atctactatgatgtatggct tgttgctaag tgcaaaacca ttcttgcatt 15300 ttccacctac tttaaatgttcttgtgtatg cagtgattgg tggcctcatt taccttcttg 15360 ctattctagt tttgaaagtggtagatgtta aagaattaaa acaaataata ggagaaaatt 15420 aggaatgaag aaagcacggaatataaactt agacttgata aaaataattg cttgtatagg 15480 agttgttttg cttcatactacgatgccagg gtttaaggaa acagggcgat ggaattactc 15540 atcttattta tattatctaggtacttatta aattaccttg ttttttatgg taaatggtta 15600 tttattattg ggtaagagcaagataacata tccctatata ctacataaaa taaaatggtt 15660 tctaataaca gtgtcttcatggaccgttat catttggttt cttaaaagag acttcacaat 15720 taatccaatt aaaaaaattttggcttcctt gatacaaaag ggttatttct tccaattttg 15780 gtttttcgga tcactaatacttatttattt atgcttgccg atattgaaga agtatttaca 15840 ttcaaaaaga agttatttatactttctata tgtattaaca attattggtt tgatttttga 15900 attgataaat tttttgcttcaaatgccagt acaaatttat gttatacaga cgtttagatt 15960 atggacttag ttcttttactacattttagg tggttttgta gcacaattca atatagagaa 16020 tttaaaatca atctttaagggatggatgaa aatagttagc atacttttgt tattgatttc 16080 accgataata ttatttttcatagcaaaaac tacttatcat aatctttttg ctgaatattt 16140 ttatgacaat cttttggtaaaagtaattag tttaggacta tttcttacct tattgacgct 16200 aaccattgat gcttctaaacatagaatgat ctacttgtta tcagtccaaa cgatgggggt 16260 atttatcata catacctatgttatgcaaat atggcaaaag ttgatagggt ttaacatagt 16320 aggtgcacac ttatttttccctgttttcac attagtgatt agttttctaa taagtatgat 16380 attaatgaaa atcccttatatcaatcgaat agttaaatta taaaaaggag tttataatgt 16440 acgattatct tattgttggtgctggtttgt ccggagcaat cttcgcacag gaagctacaa 16500 aacgtggcaa aaaagtaaaagtgattgaca agcgtgatca cattggtggc aatatctact 16560 gtgaagatgt tgaaggtattaacgttcaca agtatggtgc tcacattttc catacctcaa 16620 ataaaaaagt ttgggattatgtcaaccaat ttgctgaatt taataactat atcaactcac 16680 caattgctaa ctacaagggcagtctttata accttccatt taacatgaat acattttatg 16740 ctatgtgggg cactaagactcctcaagaag ttaaggacaa gattgctgag caaacggctg 16800 atatgaaaga tgttgagcctaaaaacttgg aagaacaagc tatcaagttg attggaccag 16860 atatctacga aaagttgatcaagggataca ctgaaaaaca atggggacgt tctgcgacag 16920 acctgcctcc tttcatcatcaagcgtcttc cggttcgtct gacttttgat aacaactact 16980 ttaatgaccg ttaccaaggaattccgatcg gtggttacaa tgtcatcatt gaaaatatgc 17040 ttggagatgt tgaagtagaacttggagttg acttctttgc caatcgtgaa gagcttgaag 17100 cttcagctga aaaagttgtctttacaggaa tgattgacca gtactttgat tataaacatg 17160 gtgagttgga gtatcgcagtcttcgttttg aacacgaagt cttggatgaa gaaaatcatc 17220 aaggaaatgc cgtggtcaactacacagagc gtgagattcc ttatactcgt atcattgagc 17280 acaagcactt cgagtatggtacacaaccta agacagttat cacacgtgaa tacccagctg 17340 attggaaacg aggagatgaaccatactacc caatcaatga tgaaaagaac aatgccatgt 17400 ttgctaagta ccaagaagaagctgagaaaa atgacaaggt tatcttctgt ggacgtcttg 17460 cagattataa atactacgacatgcacgtgg tcattgagcg tgctctagaa gtcgttgaga 17520 aagaatttac tatatgacacaagaaaaaaa ttgatatcgt tgttctttgg gtagatggaa 17580 gtgccccaga gtttatccgtgagaaacaag cagttactga gaatgtttct gatttgaacc 17640 aagaaattga tggtgagcaacgttatcgtg attatgatgt ttttaattac tggttccgaa 17700 tgattgaaaa gaatgctccttgggtaaata atgtctattt gattaccaat gggcaaaagc 17760 cagactggtt gaatttggaacatccaaaac tcaaattggt aactcatagg gaatttatgc 17820 ccaaagaata cctaccgacctataattcag cagctattga gcttaatctt catcatattg 17880 aagggttgtc ggagaactacttgtatttca atgatgatac gtacttgatt agagacagtc 17940 aaccttcaga tttttataaaaatggtcagc ctaagctttt agctgtttat gatgccttag 18000 ttccttggcc accatttacgaatacttatc acaataatgt tgaattaatt tatcgccatt 18060 ttcctaataa gaaggctttgaagtcttcgc catggaaatt ctttaatttc cgttatggtt 18120 ccttggtttt gaaaaacttgttactcttgc cttggggtcc tacgagatac gtgaaccagc 18180 atttacctgt tccgatgaagaagagtacct tggcacattt atgggaaatt gaaggtgaaa 18240 ctttagataa aacatcgcgaaatccaatta gagactatgg agtagatgtt aatcaataca 18300 tctgtcagca ttggcaaattgaaagtaacc agttttaccc tatgtctaaa agtttcggag 18360 agacaatcgg ttt 18373 5100 PRT Streptococcus macedonicus 5 Met Gly Ile Glu Ile Phe Ile Arg AsnPro Lys Gly Ile Thr Leu Thr 1 5 10 15 Lys Asp Gly Val Glu Phe Leu SerTyr Ala Arg Gln Ile Leu Glu Gln 20 25 30 Thr Ala Leu Leu Glu Glu Arg TyrLys Ser Lys Asn Thr Asn Arg Glu 35 40 45 Leu Phe Ser Val Ser Ser Gln HisTyr Ala Phe Val Val Asn Ala Phe 50 55 60 Val Ser Leu Leu Glu Gly Thr AspMet Ser Arg Tyr Glu Leu Phe Leu 65 70 75 80 Arg Glu Thr Arg Thr Tyr GluIle Ile Asp Asp Val Lys Asn Phe Arg 85 90 95 Ser Glu Ile Gly 100 6 480DNA Streptococcus macedonicus 6 tgcggttaaa gacttgcctt taagaattgtagtagttatt aaagtataca agcacaaagc 60 gcttcctttt cgagtattgc actgtatagacaaggaagat tttcgctttg ttttcgtaac 120 tgttgctttc gtatcacgac acttctatgcgatttgtcaa gagccaaaca taaaaacgag 180 aatattgcaa ggagattttc tcgacaaacaagattttaaa ccgctcgtat tctttctttg 240 agttgcggta ggaatcagtt ccattacggaaaacccaatg cttattttta aagttaaggg 300 taaagtgcga ggtttagagc atgtcgtaaactttccgaac caactcacta ttttttcgac 360 gaatcgtcgg agcaagtacg agggacaaagatgataaaat tgctatatca cattaacaac 420 ataagagtat ccgaatcaaa tcggatttttactttaaggg cgttcatctg ttatagaaga 480 7 20 DNA Streptococcus thermophilus7 atgagttcgc gtacgaatcg 20 8 20 DNA Streptococcus thermophilus 8atacagattt tagagaagcc 20 9 21 DNA Streptococcus thermophilus 9ctgcaaggcg attaagttgg g 21 10 21 DNA Streptococcus thermophilus 10gttgtgtgga attgtgagcg g 21 11 27 DNA Streptococcus macedonicus 11acaggtacct tgtctggaaa tgcagag 27 12 27 DNA Streptococcus macedonicus 12ctcggatcca accgctctat ctgctgc 27 13 27 DNA Streptococcus macedonicus 13tccggtacct ttctcttgta gtgaccg 27 14 27 DNA Streptococcus macedonicus 14cgtggatccc gtgacaaaca ctacctg 27 15 1187 DNA Streptococcus macedonicus15 tatgagttcg cgtacgaatc gtaagcaaaa gcatacgagt aatggatcgt ggggggatgg 60tcaacgttgg gttgaccatt ctgtatgcta ttttagcatt ggtcttatta ttcaccatgt 120tcaattataa tttcctatcc tttaggtttt tgaacatcat tatcaccatt ggtttgttgg 180tagttcttgc tattagcatc ttccttcaga agactaagaa atcaccacta gtgacaacgg 240ttgtactggt tatcttctcg ctagtttctc tggttggtat ttttggtttt aaacaaatga 300ttgatatcac taaccgtata aatcagactg cagccttttc agaagtagaa atgagcattg 360tggttccgaa ggatagtgac atcagagatg tgagtcagat tactagcgtt caggcaccaa 420ctaaggttga taagaataat atcgatagtt tgatgtcagc tctaaaggaa gacaaaaaag 480ttgatgacaa agttgatgat gtcgcttcct atcaagaagc ctatgacaat cttaagtctg 540gcaaatctaa agctatggtc ttgagtggct cttatgctac cctattagag tctgtcgata 600gtaattatgc ttcaaatcta aaaacaattt atacttataa aattaaaaag aaaaatagca 660actctgcaaa ccaagtagat tcaaaagtct tcaatattta tattagtggt attgatacct 720acggtccgat ttcaacagta tcacgttcag atgtcaatat cattatgaca gtaaacatga 780atacacataa gattctcttg acgactactc cacgtgatgc atacgttaag attgggcaga 840ccagtatgat aaattaaccc acgcaggtat ttatggcgtt gaaacatctg aacaaactct 900ggaagatctt tatggtatta agattgatta ctatgcacga attaacttca catctttcct 960taagttgatt gaccaacttg gtggtgtgac agtccataat gatcaagctt tcacacaagg 1020gaagtttgat ttcccggttg gagatatcca aatgaattca gagcaagcac ttggatttgt 1080tcgtgaacgc tataatttag atggcggaga taatgaccgt ggtaaaaacc aggagaaagt 1140tatttctgcg attttaaaca agttggcttc tctaaaatct gtatcaa 1187 16 1196 DNAStreptococcus thermophilus 16 tatgagttcg cgtacgaatc gtaagcaaaagcatacgagt aatggatcgt gggggatggt 60 caacgttggg ttgaccatcc tgtatgctattttagcattg gtcttattat tcaccatgtt 120 caattataat ttcctatcct ttaggtttttgaacatcatt atcaccattg gtttgttggt 180 agttcttgct attagcatct tccttcagaagactaagaaa ttaccactag tgacaacggt 240 tgtactggtt atcttctcgc tagtttctctggttggtatt tttggtttta aacaaatgat 300 tgacatcact aaccgtatga atcagacagcagcattttct gaagtagaaa tgagcatcgt 360 ggttcctaag gaaagtgaca tcaaagatgtgagccagctt actagcgtac aggcacctac 420 taaggttgat aagaacaata tcgagatcttgatgtcagct ctcaaaaaag ataaaaaagt 480 tgatgttaaa gttgatgatg ttgcctcatatcaagaagct tatgataatc tcaagtctgg 540 caaatctaaa gctatggtct tgagtggctcttatgctagc ctattagagt ctgtcgatag 600 taattatgct tcaaatctaa aaacaatttatacttataaa attaaaaaga agaatagcaa 660 ctctgcaaac caagtagatt caagagtcttcaatatttat attagtggta ttgataccta 720 cggtccgatt tcaacagtgt cacgttcagatgtcaatatc attatgacag taaacatgaa 780 tacacataag attctcttga cgactactccacgtgatgca tacgttaaga ttcctggtgg 840 tggggcagac cagtatgata aattaacccacgcaggtatt tatggcgttg aaacatctga 900 acaaactcta gaagatcttt atggtattaagcttgattac tatgcacgaa ttaacttcac 960 atctttcctt aagttgattg accaacttggtggtgtgaca gtccataatg atcaagcttt 1020 cacacaagag aagtttgatt tcccggttggagatatccaa atgaattcag agcaagcact 1080 tggatttgtt cgtgaacgct ataatttagatggcggagat aatgaccgtg gtaaaaacca 1140 ggagaaagtt atttctgcga ttttaaacaagttggcttct ctaaaatctg tatcaa 1196 17 332 PRT Streptococcus pneumoniae 17Met Ser Lys Phe Arg Asn Ile Asn Leu Asp Leu Leu Lys Val Leu Ala 1 5 1015 Cys Val Gly Val Val Leu Leu His Thr Thr Met Gly Gly Phe Lys Glu 20 2530 Thr Gly Ala Trp Asn Phe Leu Thr Tyr Leu Tyr Tyr Leu Gly Thr Tyr 35 4045 Ser Ile Pro Leu Phe Phe Met Val Asn Gly Tyr Leu Leu Leu Gly Lys 50 5560 Arg Glu Ile Thr Tyr Ser Tyr Ile Leu Gln Lys Ile Lys Trp Leu Leu 65 7075 80 Ile Thr Val Ser Ser Trp Thr Phe Ile Val Trp Leu Phe Lys Arg Asp 8590 95 Phe Thr Glu Asn Leu Ile Lys Lys Ile Ile Gly Ser Leu Ile Gln Lys100 105 110 Gly Tyr Phe Phe Gln Phe Trp Phe Phe Gly Ala Leu Ile Leu IleTyr 115 120 125 Leu Cys Leu Pro Ile Leu Arg Gln Phe Leu Asn Ser Lys ArgSer Tyr 130 135 140 Leu Tyr Ser Leu Ser Leu Leu Met Thr Ile Gly Leu IlePhe Glu Leu 145 150 155 160 Ser Asn Ile Leu Leu Gln Met Pro Ile Gln ThrTyr Val Ile Gln Thr 165 170 175 Phe Arg Leu Trp Thr Trp Phe Phe Tyr TyrLeu Leu Gly Gly Tyr Ile 180 185 190 Ala Gln Phe Thr Ile Glu Glu Ile GluSer Arg Phe Lys Asn Trp Met 195 200 205 Lys Ile Val Ser Ile Leu Leu LeuLeu Ile Ser Pro Ile Ile Leu Phe 210 215 220 Phe Ile Ala Lys Thr Ile TyrHis Asn Leu Phe Ala Glu Tyr Phe Tyr 225 230 235 240 Asp Thr Leu Phe ValLys Val Ser Thr Leu Gly Ile Phe Leu Thr Ile 245 250 255 Leu Met Leu ThrLeu Asn Glu Asn Arg Arg Glu Ser Ile Val Ser Leu 260 265 270 Ser Asn GlnThr Met Gly Val Phe Ile Ile His Thr Tyr Ile Met Lys 275 280 285 Val TrpGlu Lys Val Leu Gly Phe Asn Phe Val Gly Ala Tyr Leu Leu 290 295 300 PheAla Leu Phe Thr Leu Ser Val Ser Phe Ile Ile Val Gly Met Leu 305 310 315320 Met Lys Ile Pro Tyr Phe Asn Arg Ile Val Lys Leu 325 330 18 493 PRTStreptococcus macedonicus 18 Met Ser Lys His Ser Arg His Arg Arg His HisLys Ser Ser Arg Ser 1 5 10 15 Tyr Ser Arg Phe Asp Thr Lys Thr Ile ValAsn Ser Val Leu Leu Val 20 25 30 Leu Phe Ala Leu Leu Ala Gly Ile Ala ThrTyr Leu Met Tyr Ala Asn 35 40 45 Asn Ile Leu Ala Phe Arg His Leu Asn IleIle Tyr Thr Val Leu Leu 50 55 60 Val Ala Val Phe Leu Ile Ser Leu Val LeuIle Ile Arg Lys Lys Gly 65 70 75 80 Lys Ile Val Val Thr Val Leu Leu ValIle Phe Ser Ile Val Ala Ala 85 90 95 Ile Ser Leu Phe Ala Phe Lys Ser LeuVal Asp Val Ala Asn Asp Met 100 105 110 Asn Lys Ser Ala Ser Tyr Ser GluIle Glu Met Ser Val Val Val Pro 115 120 125 Ala Asp Ser Ser Ile Ser AspVal Thr Glu Leu Ser Ser Val Gln Ala 130 135 140 Pro Thr Asn Ala Asp GlySer Asn Ile Asp Thr Leu Leu Ser Gln Ile 145 150 155 160 Lys Ser Asp LysGly Ile Asp Leu Ala Thr Glu Thr Val Asp Ser Tyr 165 170 175 Gln Ala AlaTyr Glu Asn Leu Ile Asn Gly Ser Ser Gln Ala Met Val 180 185 190 Leu AsnSer Ala Tyr Ser Ser Leu Leu Glu Leu Ser Tyr Asn Asp Tyr 195 200 205 GluSer Asn Leu Lys Thr Ile Tyr Thr Tyr Lys Ile Lys Lys Ser Val 210 215 220Ser Ser Glu Ala Lys Ser Ser Asp Ala Asn Val Phe Asn Ile Tyr Ile 225 230235 240 Ser Gly Ile Asp Thr Tyr Gly Ser Ile Ser Thr Val Ser Arg Ser Asp245 250 255 Val Asn Ile Ile Leu Thr Val Asn Met Asn Thr His Lys Ile LeuMet 260 265 270 Thr Thr Ala Pro Arg Asp Ser Tyr Val Gln Ile Pro Asp GlyGly Ala 275 280 285 Asp Gln Tyr Asp Lys Leu Thr His Ala Gly Ile Tyr GlyVal Glu Thr 290 295 300 Ser Glu Lys Thr Leu Glu Asn Leu Tyr Gly Ile AspIle Asp Tyr Tyr 305 310 315 320 Ala Arg Ile Asn Phe Thr Ser Phe Met AsnLeu Ile Asp Ala Ile Gly 325 330 335 Gly Val Thr Val Tyr Asn Asp Gln AlaPhe Thr Ser Leu His Gly Asn 340 345 350 Tyr Asn Phe Glu Val Gly Asn ValAsn Leu Ser Ser Gly Glu Glu Ala 355 360 365 Leu Ala Phe Val Arg Glu ArgTyr Ser Leu Asn Asn Gly Asp Tyr Asp 370 375 380 Arg Gly Asn Asn Gln IleLys Val Ile Gln Ala Ile Val Asn Lys Leu 385 390 395 400 Thr Ser Leu SerSer Ile Ser Asn Tyr Ser Thr Ile Ile Ser Thr Leu 405 410 415 Gln Asp SerIle Gln Thr Asp Met Ser Leu Asp Thr Met Met Ser Leu 420 425 430 Ala AsnAla Gln Leu Asp Ser Gly Lys Lys Phe Thr Ile Thr Ser Gln 435 440 445 GluVal Thr Gly Thr Gly Ser Thr Gly Glu Leu Thr Ser Tyr Ala Met 450 455 460Pro Thr Ala Ser Leu Tyr Met Ile Gln Leu Asp Asp Ala Ser Val Ala 465 470475 480 Ser Ala Ser Gln Ala Ile Lys Asp Val Met Glu Gly Lys 485 490 19243 PRT Streptococcus macedonicus 19 Met Ile Asp Ile His Ser His Ile ValPhe Asp Val Asp Asp Gly Pro 1 5 10 15 Thr Thr Ile Glu Glu Ser Leu AlaLeu Val Gly Glu Ser Tyr Arg Gln 20 25 30 Gly Val Arg Thr Ile Val Ser ThrSer His Arg Arg Lys Gly Met Phe 35 40 45 Glu Thr Pro Glu Asp Lys Ile PheAla Asn Phe Ser Gln Val Lys Glu 50 55 60 Ala Ala Glu Ala Lys Tyr Glu GlyLeu Glu Ile Leu Tyr Gly Gly Glu 65 70 75 80 Leu Tyr Tyr Ser Ser Asp IleLeu Glu Arg Leu Glu Gln Arg Gln Val 85 90 95 Pro Arg Met Asn Asp Thr ArgPhe Ala Leu Ile Glu Phe Ser Met Thr 100 105 110 Thr Pro Trp Lys Glu IleHis Thr Ala Leu Ser Asn Val Ile Met Leu 115 120 125 Gly Ile Thr Pro ValVal Ala His Ile Glu Arg Tyr Asn Ala Leu Glu 130 135 140 Phe Asn Glu GluArg Val Lys Glu Leu Ile Asn Met Gly Gly Tyr Thr 145 150 155 160 Gln IleAsn Ser Ser His Val Leu Lys Pro Lys Leu Phe Gly Asp Lys 165 170 175 TyrHis Gln Phe Lys Lys Arg Ala Arg Tyr Phe Leu Glu Lys Asn Leu 180 185 190Val His Cys Val Ala Ser Asp Met His Asn Leu Gly Pro Arg Pro Pro 195 200205 Phe Met Asp Lys Ala Arg Glu Ile Val Thr Lys Asp Phe Gly Pro Asn 210215 220 Arg Ala Tyr Ala Leu Phe Glu Glu Asn Pro Gln Thr Leu Leu Glu Asn225 230 235 240 Lys Asp Leu 20 229 PRT Streptococcus macedonicus 20 MetAsn Ser Asn Asp Asn Ala Ser Ile Glu Ile Asp Val Leu Tyr Leu 1 5 10 15Leu Arg Lys Leu Trp Ser Arg Lys Phe Phe Ile Ile Phe Ile Ala Leu 20 25 30Val Val Gly Thr Val Ala Leu Leu Gly Ser Val Phe Phe Leu Lys Pro 35 40 45Lys Tyr Thr Ser Thr Thr Arg Ile Tyr Val Val Ser Arg Ser Ser Asp 50 55 60Gly Ser Leu Thr Asn Gln Asp Leu Gln Ala Gly Ser Tyr Leu Val Asn 65 70 7580 Asp Tyr Lys Glu Val Ile Thr Ser Asn Glu Val Leu Ser Ser Val Ile 85 9095 Ser Gln Glu Asn Leu Ser Leu Ser Thr Ser Glu Leu Ser Asn Met Ile 100105 110 Ser Val Asn Ile Pro Thr Asp Thr Arg Val Ile Ser Ile Ser Val Glu115 120 125 Asp Thr Asp Ala Lys Glu Ala Ser Asp Ile Ala Asn Thr Ile ArgGlu 130 135 140 Val Ala Ala Glu Lys Ile Lys Ser Val Thr Lys Val Asp AspVal Thr 145 150 155 160 Thr Leu Glu Ala Ala Glu Val Ala Ser Lys Pro SerSer Pro Asn Ile 165 170 175 Lys Arg Asn Ala Ala Leu Gly Val Leu Val GlyGly Phe Leu Ala Ile 180 185 190 Val Gly Ile Leu Val Leu Glu Val Leu AspAsp Arg Val Arg Arg Pro 195 200 205 Glu Asp Val Glu Glu Val Leu Gly MetThr Leu Leu Gly Val Val Pro 210 215 220 Asp Ile Asp Lys Leu 225 21 213PRT Streptococcus macedonicus 21 Met Pro Gln Leu Glu Leu Val Arg Ala LysAla Gln Met Val Lys Ser 1 5 10 15 Met Glu Glu Tyr Tyr Asn Ser Ile ArgThr Asn Ile Gln Phe Ser Gly 20 25 30 Arg Asp Leu Lys Val Ile Thr Leu ThrSer Ala Gln Ser Gly Glu Gly 35 40 45 Lys Ser Thr Thr Ser Val Asn Leu AlaIle Ser Phe Ala Arg Ala Gly 50 55 60 Phe Arg Thr Leu Leu Ile Asp Ala AspThr Arg Asn Ser Val Met Ser 65 70 75 80 Gly Thr Phe Lys Ser Lys Glu ArgTyr Gln Gly Leu Thr Ser Phe Leu 85 90 95 Ser Gly Asn Ala Glu Leu Ser AspVal Ile Cys Asp Thr Asn Ile Asp 100 105 110 Asn Leu Met Ile Ile Pro AlaGly Gln Val Pro Pro Asn Pro Thr Ser 115 120 125 Leu Ile Gln Asn Asp AsnPhe Lys Ala Met Ile Glu Ile Ile Arg Gly 130 135 140 Leu Tyr Asp Tyr ValIle Ile Asp Thr Pro Pro Leu Gly Leu Val Ile 145 150 155 160 Asp Ala AlaIle Leu Ala His Tyr Ser Asp Ala Ser Leu Leu Val Val 165 170 175 Lys AlaGly Ala Asp Lys Arg Arg Thr Val Thr Lys Leu Lys Glu Gln 180 185 190 LeuGlu Gln Ser Gly Ser Ala Phe Leu Gly Val Ile Leu Asn Lys Tyr 195 200 205Asp Ile Gln Val Val 210 22 458 PRT Streptococcus macedonicus 22 Met TyrSer Glu Asp Ser Lys Lys Lys Val Tyr Tyr Leu Leu Ser Asp 1 5 10 15 IleIle Ala Leu Val Ile Ser Tyr Leu Ile Leu Ala Gln Phe Tyr Pro 20 25 30 TyrHis Phe Phe Asp Ser Lys Phe Phe Ala Val Val Phe Gly Ile Leu 35 40 45 IleVal Ile Val Ser Val Leu Ser Asp Glu Tyr Ser Ser Ile Lys Asn 50 55 60 ArgGly Tyr Leu Lys Glu Leu Lys Ala Ser Val Ile Tyr Gly Met Lys 65 70 75 80Val Leu Val Leu Phe Thr Phe Val Leu Ile Leu Gly Lys Ile Arg Phe 85 90 95Ile His Asp Ile Ser Gln Met Ser Tyr Phe Leu Leu Gly Gln Ile Phe 100 105110 Ile Leu Val Ser Leu Phe Val Phe Ile Gly Arg Ile Leu Val Lys Asn 115120 125 Leu Phe Arg Ser His Ala Thr Asp Ile Lys Gln Val Val Phe Val Thr130 135 140 Asp Phe Thr Asn Gly Lys Glu Val Ile Lys Glu Leu Ser Asn SerAsn 145 150 155 160 Tyr His Ile Ala Ala Tyr Ile Ser Arg Arg Asp Asn ProAsp Ile Ser 165 170 175 Gln Pro Ile Leu Lys Ser Thr Lys Glu Ile Arg AspPhe Val Ala Asn 180 185 190 His Gln Val Asp Glu Ile Phe Val Ala Lys AsnHis Gln Asp Asp Phe 195 200 205 Ile Glu Phe Ala His Cys Leu Lys Leu LeuGly Ile Pro Thr Thr Val 210 215 220 Ala Val Gly Asn Tyr Ser Asp Phe TyrVal Gly Asn Ser Val Leu Lys 225 230 235 240 Lys Val Gly Asp Thr Thr PheIle Thr Thr Ala Phe Asn Ile Val Lys 245 250 255 Phe Arg Gln Ile Ala LeuLys Arg Leu Met Asp Ile Ala Ile Ala Leu 260 265 270 Val Gly Leu Val IleThr Gly Ile Val Ala Ile Ile Ile Thr Pro Ile 275 280 285 Ile Lys Lys GlnSer Pro Gly Pro Leu Ile Phe Lys Gln Lys Arg Val 290 295 300 Gly Lys AsnGly Lys Val Phe Glu Ile Tyr Lys Phe Arg Ser Met Tyr 305 310 315 320 ThrAsp Ala Glu Glu Arg Lys Lys Glu Leu Leu Thr Gln Asn Asp Leu 325 330 335Asp Thr Asp Leu Met Phe Lys Met Asp Asp Asp Pro Arg Ile Phe Pro 340 345350 Phe Gly His Lys Leu Arg Asp Trp Ser Leu Asp Glu Leu Pro Gln Phe 355360 365 Ile Asn Val Leu Lys Gly Glu Met Ser Val Val Gly Thr Arg Pro Pro370 375 380 Thr Leu Asp Glu Tyr His His Tyr Glu Leu His His Phe Lys ArgLeu 385 390 395 400 Thr Thr Lys Pro Gly Ile Thr Gly Leu Trp Gln Val SerGly Arg Ser 405 410 415 Asp Ile Thr Asp Phe Glu Glu Val Val Ala Leu AspMet Lys Tyr Ile 420 425 430 Gln Asn Trp Ser Ile Ser Glu Asp Ile Lys IleIle Ala Lys Thr Phe 435 440 445 Gly Val Val Leu Lys Arg Glu Gly Ser Lys450 455 23 149 PRT Streptococcus macedonicus 23 Met Lys Val Cys Leu ValGly Ser Ser Gly Gly His Leu Ala His Leu 1 5 10 15 Asn Met Leu Lys ProPhe Trp Ser Glu His Ser Arg Phe Arg Val Thr 20 25 30 Phe Asp Lys Glu AspAla Arg Ser Val Leu Ser Asp Glu Lys Phe Tyr 35 40 45 Pro Cys Tyr Phe ProThr Asn Arg Asn Phe Lys Asn Leu Val Lys Asn 50 55 60 Thr Phe Leu Ala LeuGlu Ile Leu Arg Lys Glu Lys Pro Asp Val Ile 65 70 75 80 Ile Ser Ser GlyAla Ala Val Ala Val Pro Phe Phe Tyr Leu Gly Lys 85 90 95 Leu Phe Gly AlaLys Thr Val Tyr Ile Glu Val Phe Asp Arg Ile Asp 100 105 110 Lys Pro ThrVal Thr Gly Lys Leu Val Tyr Pro Val Thr Asp Lys Phe 115 120 125 Ile ValGln Trp Glu Glu Met Lys Thr Val Tyr Pro Lys Ala Ile Asn 130 135 140 LeuGly Ser Ile Phe 145 24 161 PRT Streptococcus macedonicus 24 Met Ile PheVal Thr Val Gly Thr His Glu Gln Pro Phe Asn Arg Leu 1 5 10 15 Ile LysGlu Val Asp Arg Leu Lys Lys Glu Gly Ile Ile Thr Asp Glu 20 25 30 Val PheIle Gln Thr Gly Phe Ser Thr Tyr Glu Pro Gln Tyr Cys Asp 35 40 45 Trp LysAsn Ile Ile Ser Tyr Ser Glu Met Glu Asp Tyr Met Asn Arg 50 55 60 Ala AspIle Ile Ile Thr His Gly Gly Pro Ala Thr Phe Met Gly Ala 65 70 75 80 IleAla Lys Gly Lys Lys Pro Ile Val Val Pro Arg Gln Glu Lys Phe 85 90 95 GlyGlu His Val Asn Asp His Gln Leu Glu Phe Ala Glu Gln Val Ser 100 105 110Glu Arg Phe Gly Ser Ile Val Val Val Glu Glu Ile Asn Glu Leu Gln 115 120125 Asn Tyr Phe Asn Leu Asp Leu Ile Val Asp Glu Ser Ser Asn Ser Asn 130135 140 Asn Leu Arg Phe Asn Ser Gln Leu Lys Gln Glu Ile Glu Ser Leu Val145 150 155 160 Arg 25 245 PRT Streptococcus macedonicus 25 Met Ile ProLys Lys Ile His Tyr Cys Trp Phe Gly Gly Asn Pro Leu 1 5 10 15 Pro AspSer Val Lys Asn Cys Ile Asn Ser Trp Lys Lys Phe Cys Pro 20 25 30 Asn TyrGlu Ile Ile Glu Trp Asn Glu Ser Asn Tyr Asp Val His Lys 35 40 45 Ile ProTyr Ile Ser Glu Ala Tyr Lys Asn Lys Lys Tyr Ala Phe Val 50 55 60 Ser AspTyr Ala Arg Leu Asp Ile Ile Tyr Asn Glu Gly Gly Phe Tyr 65 70 75 80 LeuAsp Thr Asp Val Glu Leu Leu Lys Ala Leu Asp Asp Leu Thr Ser 85 90 95 GluHis Cys Tyr Met Gly Met Glu Gln Val Gly Arg Val Asn Thr Gly 100 105 110Leu Gly Phe Gly Ala Glu Lys Gly His Leu Phe Ile Lys Glu Asn Met 115 120125 Gln Gln Tyr Glu Glu Val Ser Phe Asn Leu Lys Leu Leu Glu Thr Cys 130135 140 Val Asp Ile Thr Thr Asn Leu Leu Leu Ser Lys Gly Leu Leu Val Glu145 150 155 160 Asn Ser Tyr Gln Lys Ile Ser Asp Val Ser Ile Tyr Pro ThrAsp Phe 165 170 175 Phe Cys Pro Phe Asn Met Gln Thr Gln Glu Met Gly IleThr Lys Asn 180 185 190 Thr Tyr Ser Ile His His Tyr Asp Ser Thr Trp TyrGly Asn Gly Val 195 200 205 Ser Ala Ile Ile Lys Lys Lys Leu Leu Pro LeuArg Val Lys Ser Arg 210 215 220 Ile Leu Ile Asp Lys Tyr Leu Gly Glu GlySer Tyr Ala Lys Ile Lys 225 230 235 240 Ala Ile Ile Lys Lys 245 26 249PRT Streptococcus macedonicus 26 Met Val Ser Leu Ser Lys Leu Asn Ile IleLys Asp Asn Leu Phe Leu 1 5 10 15 Phe Tyr Arg Asp Gly Gln Phe Val GlyArg His Thr Phe Gly Tyr Gly 20 25 30 His Pro Asn Gln Ala Gln Ser Ala LeuThr Ile Leu Ile Ile Leu Ala 35 40 45 Ile Tyr Leu Tyr Asn Glu Lys Phe AsnIle Phe His Tyr Ile Ile Met 50 55 60 Ile Ile Met Asn Phe Tyr Leu Tyr SerLeu Thr Tyr Ser Arg Thr Gly 65 70 75 80 Phe Leu Ile Gly Val Leu Cys IleVal Leu Gly Val Val Gln Lys Ser 85 90 95 Lys Asn Val Glu Lys Ile Phe AlaArg Val Phe Lys Asn Ser Tyr Phe 100 105 110 Trp Ala Val Leu Val Thr LeuPhe Ile Gly Tyr Phe Tyr Thr Lys Ile 115 120 125 Pro Gln Leu Lys Asn LeuAsp Glu Leu Phe Thr Gly Arg Leu Ala Tyr 130 135 140 Asn Asn Thr Leu LeuAsn Asn Tyr Ile Pro Pro Leu Ile Gly Ser Ser 145 150 155 160 Lys Tyr AsnGlu Tyr Val Asn Ile Asp Asn Gly Phe Ile Ser Leu Ile 165 170 175 Tyr GlnGly Gly Ile Leu Ala Phe Leu Trp Ile Ser Ala Cys Ile Ile 180 185 190 LysLeu Met Asn Asp Phe Tyr Ile Gln Lys Lys Phe Arg Glu Leu Phe 195 200 205Phe Met Ser Ser Phe Ile Val Tyr Gly Met Thr Glu Ser Phe Phe Pro 210 215220 Asn Ile Ala Val Asn Ile Ser Leu Ile Phe Ile Gly Lys Leu Ile Phe 225230 235 240 Lys Thr Arg Glu Glu Val Met Asn Ala 245 27 292 PRTStreptococcus macedonicus 27 Met His Lys Val Phe Ile Phe Thr Pro Thr TyrAsn Arg Val Glu Asn 1 5 10 15 Leu Lys Lys Leu Tyr Glu Ser Leu Arg LysGln Thr Cys Lys Glu Phe 20 25 30 Ile Trp Leu Ile Val Asp Asp Gly Ser AsnAsp Gly Thr Glu Phe Tyr 35 40 45 Ile Arg Gln Leu Arg Ser Glu Tyr Ile PheAsp Ile Val Tyr Leu Lys 50 55 60 Lys Glu Asn Gly Gly Lys His Thr Ala TyrAsn Leu Ala Leu Asp Tyr 65 70 75 80 Met Gly Gly Glu Gly Trp His Met ValVal Asp Ser Asp Asp Trp Leu 85 90 95 Ala Ser Thr Ala Val Glu Cys Ile IleLys Asp Ile Ser Ser Leu Gln 100 105 110 Val Gly Lys Leu Gly Val Val TyrPro Lys Tyr Ser Leu Thr Glu Glu 115 120 125 Leu Arg Trp Leu Pro Glu LysVal Thr Glu Val Asn Ile Pro Asp Ile 130 135 140 Lys Leu Lys Tyr Gly LeuSer Ile Glu Thr Ala Ile Val Ile Lys Asn 145 150 155 160 Leu Phe Ile GlyGln Leu Arg Leu Pro Ser Phe Glu Gly Glu Lys Phe 165 170 175 Leu Ser GluGlu Ile Phe Tyr Ile Met Leu Ser Glu Phe Gly Lys Phe 180 185 190 Leu ProLeu Asn Arg Arg Ile Tyr Phe Phe Glu Tyr Leu Glu His Gly 195 200 205 LeuThr Asn Asn Leu Phe His Leu Trp Lys Lys Asn Pro Lys Ser Thr 210 215 220Tyr Leu Leu Phe Lys Glu Arg Lys Lys Tyr Ile Leu Gln Asn Leu Ser 225 230235 240 Gly Phe Asn Arg Ile Val Glu Leu Phe Lys Val Ser Leu Asn Glu Gln245 250 255 Ala Leu Ser Leu Ala Thr Ser Lys Asn Glu Asn Ile Pro Gln GluLeu 260 265 270 Ser Val Gly Glu Arg Met Leu Lys Pro Leu Ala Tyr Leu PheTyr Leu 275 280 285 Lys Arg Tyr Lys 290 28 320 PRT Streptococcusmacedonicus 28 Val Asp Asn Glu Leu Ile Ser Ile Ile Val Pro Val Tyr AsnVal Glu 1 5 10 15 Lys Tyr Ile Ala Lys Cys Leu Asp Ser Leu Val Asn GlnThr Tyr Leu 20 25 30 Asn Ile Glu Ile Leu Leu Ile Asp Asp Gly Ser Thr AspLys Ser Leu 35 40 45 Ser Ile Cys Lys Lys Tyr Ala Ala Val Asp Ser Arg IleLys Leu Phe 50 55 60 Ser Lys Glu Asn Gly Gly Val Ser Ser Ala Arg Asn LeuGly Leu Leu 65 70 75 80 His Val Gln Gly Glu Tyr Val Val Phe Val Asp SerAsp Asp Phe Val 85 90 95 Ser Pro Lys Tyr Cys Glu His Leu Tyr Gln Leu ThrIle Ser Thr Lys 100 105 110 Ser Glu Leu Ala Ser Val Ser Arg Tyr Asn IleLeu Asn Lys Glu Val 115 120 125 Val Lys Ile Ser Asp Leu Ser Phe Asn GlnIle Thr Ser Asp Glu Ala 130 135 140 Leu Arg Lys Phe Phe Leu Gly Glu GlyIle Asn Cys Tyr Leu Phe Ser 145 150 155 160 Lys Ile Phe Lys Tyr Glu ThrIle Lys Gly Leu Arg Phe Asp Glu Ser 165 170 175 Leu Glu Ser Ala Glu AspVal Leu Phe Ile Tyr Gln Thr Leu Lys Asn 180 185 190 Ile Asn Phe Ala SerMet Asp Gly Thr Val Ala Asp Tyr Phe Tyr Ile 195 200 205 Leu Arg Glu GlySer Leu Thr Asn Lys Arg Leu Thr Ser Ser Arg Ile 210 215 220 Asp Ser SerIle Arg Val Ala Glu Phe Ile Thr Arg Asp Cys Asn Ser 225 230 235 240 AsnLys Lys Leu Lys Met Leu Ser Glu Ile Asn Glu Ile Ser Leu Lys 245 250 255Gly Glu Val Leu Glu Trp Ile Ser Leu Asn Ser Glu Leu Arg Ile Glu 260 265270 Phe Glu Glu Tyr Tyr Asn Ile Ile Leu Arg Glu Val Arg Lys Phe Lys 275280 285 Leu Leu His Lys Val Gln Tyr Leu Thr Leu Lys Lys Phe Ile Arg Ile290 295 300 Ile Leu Leu Lys Val Ser Pro Arg Leu Val Thr Ile Leu Lys AsnLys 305 310 315 320 29 271 PRT Streptococcus macedonicus 29 Met Asp ArgGly Gly Met Val Asn Val Gly Leu Thr Ile Leu Tyr Ala 1 5 10 15 Ile LeuAla Leu Val Leu Leu Phe Thr Met Phe Asn Tyr Asn Phe Leu 20 25 30 Ser PheArg Phe Leu Asn Ile Ile Ile Thr Ile Gly Leu Leu Val Val 35 40 45 Leu AlaIle Ser Ile Phe Leu Gln Lys Thr Lys Lys Ser Pro Leu Val 50 55 60 Thr ThrVal Val Leu Val Ile Phe Ser Leu Val Ser Leu Val Gly Ile 65 70 75 80 PheGly Phe Lys Gln Met Ile Asp Ile Thr Asn Arg Ile Asn Gln Thr 85 90 95 AlaAla Phe Ser Glu Val Glu Met Ser Ile Val Val Pro Lys Asp Ser 100 105 110Asp Ile Arg Asp Val Ser Gln Ile Thr Ser Val Gln Ala Pro Thr Lys 115 120125 Val Asp Lys Asn Asn Ile Asp Ser Leu Met Ser Ala Leu Lys Glu Asp 130135 140 Lys Lys Val Asp Asp Lys Val Asp Asp Val Ala Ser Tyr Gln Glu Ala145 150 155 160 Tyr Asp Asn Leu Lys Ser Gly Lys Ser Lys Ala Met Val LeuSer Gly 165 170 175 Ser Tyr Ala Thr Leu Leu Glu Ser Val Asp Ser Asn TyrAla Ser Asn 180 185 190 Leu Lys Thr Ile Tyr Thr Tyr Lys Ile Lys Lys LysAsn Ser Asn Ser 195 200 205 Ala Asn Gln Val Asp Ser Lys Val Phe Asn IleTyr Ile Ser Gly Ile 210 215 220 Asp Thr Tyr Gly Pro Ile Ser Thr Val SerArg Ser Asp Val Asn Ile 225 230 235 240 Ile Met Thr Val Asn Met Asn ThrHis Lys Ile Leu Leu Thr Thr Thr 245 250 255 Pro Arg Asp Ala Tyr Val LysIle Gly Gln Thr Ser Met Ile Asn 260 265 270 30 131 PRT Streptococcusmacedonicus 30 Met Asn Ser Glu Gln Ala Leu Gly Phe Val Arg Glu Arg TyrAsn Leu 1 5 10 15 Asp Gly Gly Asp Asn Asp Arg Gly Lys Asn Gln Glu LysVal Ile Ser 20 25 30 Ala Ile Leu Asn Lys Leu Ala Ser Leu Lys Ser Val SerAsn Phe Thr 35 40 45 Ser Ile Val Asn Asn Leu Gln Asp Ser Val Gln Thr AsnMet Ser Leu 50 55 60 Asn Pro Ile Asn Ala Leu Ala Asn Thr Gln Leu Glu SerGly Ser Lys 65 70 75 80 Phe Thr Val Thr Ser Gln Ala Val Thr Gly Thr GlySer Thr Gly Gln 85 90 95 Leu Thr Ser Tyr Ala Met Pro Asn Ser Ser Leu TyrMet Met Lys Leu 100 105 110 Asp Asn Ser Ser Val Glu Ser Ala Ser Gln AlaIle Lys Lys Leu Met 115 120 125 Glu Glu Lys 130 31 162 PRT Streptococcusmacedonicus 31 Val Ile Asp Val His Ser His Ile Val Phe Asp Val Asp AspGly Pro 1 5 10 15 Lys Thr Leu Glu Glu Ser Leu Asp Leu Ile Gly Glu SerTyr Ala Gln 20 25 30 Gly Val Arg Lys Ile Val Ser Thr Ser His Arg Arg LysGly Met Phe 35 40 45 Glu Thr Pro Glu Asn Lys Ile Phe Ala Asn Phe Ser LysVal Lys Ala 50 55 60 Glu Ala Glu Ala Leu Tyr Pro Asp Leu Thr Ile Tyr TyrGly Gly Glu 65 70 75 80 Leu Asp Tyr Thr Leu Asp Ile Val Glu Lys Leu GluLys Asn Leu Ile 85 90 95 Pro Arg Met His Asn Thr Gln Phe Ala Leu Ile GluPhe Ser Ala Arg 100 105 110 Thr Ser Trp Lys Glu Ile His Ser Gly Leu SerAsn Val Leu Arg Ala 115 120 125 Gly Val Thr Pro Ile Val Ala His Ile GluArg Tyr Asp Ala Leu Glu 130 135 140 Glu Asn Ala Asp Arg Val Arg Glu IleIle Asn Tyr Asp Thr Arg Asn 145 150 155 160 Cys Lys 32 471 PRTStreptococcus macedonicus 32 Met Lys Val Leu Lys Asn Tyr Ala Tyr Asn LeuSer Tyr Gln Leu Leu 1 5 10 15 Val Ile Val Leu Pro Ile Ile Thr Thr ProTyr Val Thr Arg Ile Phe 20 25 30 Ser Ser Lys Asp Leu Gly Thr Tyr Gly TyrPhe Asn Ser Ile Val Ala 35 40 45 Tyr Phe Ile Leu Leu Ala Thr Leu Gly ValAla Asn Tyr Gly Thr Lys 50 55 60 Glu Ile Ser Gly His Arg Lys Asp Ile ArgLys Asn Phe Trp Gly Ile 65 70 75 80 Tyr Thr Leu Gln Leu Ile Ala Thr IleLeu Ser Leu Val Leu Tyr Thr 85 90 95 Ser Leu Cys Leu Phe Phe Pro Gly MetGln Asn Met Val Ala Tyr Ile 100 105 110 Leu Gly Leu Ser Leu Ile Ser LysGly Met Asp Ile Ser Trp Leu Phe 115 120 125 Gln Gly Leu Glu Asp Phe ArgArg Ile Thr Ala Arg Asn Thr Thr Val 130 135 140 Lys Val Leu Gly Val IleSer Ile Phe Leu Phe Val Lys Thr Pro Gly 145 150 155 160 Asp Leu Tyr LeuTyr Val Phe Leu Leu Thr Phe Phe Glu Leu Leu Gly 165 170 175 Gln Leu SerMet Trp Leu Pro Ala Arg Pro Tyr Ile Gly Lys Pro Gln 180 185 190 Phe AspLeu Ser Tyr Ala Lys Lys Arg Leu Lys Pro Val Ile Leu Leu 195 200 205 PheLeu Pro Gln Val Ala Ile Ser Leu Tyr Val Thr Leu Asp Arg Thr 210 215 220Met Leu Gly Ala Leu Ser Ser Thr Asn Asp Val Gly Ile Tyr Asp Gln 225 230235 240 Ala Leu Lys Ile Ile Asn Ile Leu Leu Thr Leu Val Thr Ser Leu Gly245 250 255 Ser Val Met Leu Pro Arg Val Ser Gly Leu Leu Ser Asn Gly AspHis 260 265 270 Lys Ala Val Asn Lys Met His Glu Leu Ser Phe Leu Ile TyrAsn Leu 275 280 285 Val Ile Phe Pro Ile Ile Ala Gly Leu Leu Ile Val AsnLys Asp Phe 290 295 300 Val Ser Phe Phe Leu Gly Lys Asp Phe Gln Glu AlaTyr Leu Ala Ile 305 310 315 320 Ala Ile Met Val Phe Arg Met Phe Phe IleGly Trp Thr Asn Ile Met 325 330 335 Gly Ile Gln Ile Leu Ile Pro His AsnLys His Arg Glu Phe Met Leu 340 345 350 Ser Thr Thr Ile Pro Ala Val ValSer Val Gly Leu Asn Leu Leu Leu 355 360 365 Ile Pro Pro Phe Gly Phe ValGly Ala Ser Ile Val Ser Val Leu Thr 370 375 380 Glu Ala Leu Val Trp PheIle Gln Leu Tyr Phe Cys Leu Pro Tyr Leu 385 390 395 400 Lys Glu Val ProIle Leu Glu Ser Leu Ala Lys Ile Val Cys Ala Ser 405 410 415 Thr Met MetTyr Gly Leu Leu Leu Ser Ala Lys Pro Phe Leu His Phe 420 425 430 Pro ProThr Leu Asn Val Leu Val Tyr Ala Val Ile Gly Gly Leu Ile 435 440 445 TyrLeu Leu Ala Ile Leu Val Leu Lys Val Val Asp Val Lys Glu Leu 450 455 460Lys Gln Ile Ile Gly Glu Asn 465 470 33 332 PRT Streptococcus macedonicusmisc_feature (49)..(49) Xaa can be any naturally occurring amino acid 33Met Lys Lys Ala Arg Asn Ile Asn Leu Ser Leu Ile Leu Ile Ile Gly 1 5 1015 Cys Ile Gly Val Val Leu Leu His Thr Thr Met Pro Gly Phe Leu Glu 20 2530 Thr Gly Arg Trp Asn Tyr Ser Ser Tyr Leu Tyr Tyr Leu Gly Thr Tyr 35 4045 Xaa Ile Thr Leu Phe Phe Met Val Asn Gly Tyr Leu Leu Leu Gly Lys 50 5560 Ser Lys Ile Thr Tyr Pro Tyr Ile Leu His Lys Ile Lys Trp Phe Leu 65 7075 80 Ile Thr Val Ser Ser Trp Thr Val Ile Ile Trp Phe Leu Lys Arg Asp 8590 95 Phe Thr Ile Asn Pro Ile Lys Lys Ile Leu Ala Ser Leu Ile Gln Lys100 105 110 Gly Tyr Phe Phe Gln Phe Trp Phe Phe Gly Ser Leu Ile Leu IleTyr 115 120 125 Leu Cys Leu Pro Ile Leu Lys Lys Tyr Leu His Ser Lys ArgSer Tyr 130 135 140 Leu Tyr Phe Leu Tyr Val Leu Thr Ile Ile Gly Leu IlePhe Glu Leu 145 150 155 160 Ile Asn Phe Leu Leu Gln Met Pro Val Gln IleTyr Val Ile Gln Thr 165 170 175 Phe Arg Leu Trp Thr Xaa Phe Phe Tyr TyrIle Leu Gly Gly Phe Val 180 185 190 Ala Gln Phe Ile Ile Glu Asn Leu LysSer Ile Phe Leu Gly Trp Met 195 200 205 Lys Ile Val Ser Ile Leu Leu LeuLeu Ile Ser Pro Ile Ile Leu Phe 210 215 220 Phe Ile Ala Lys Thr Thr TyrHis Asn Leu Phe Ala Glu Tyr Phe Tyr 225 230 235 240 Asp Asn Leu Leu ValLys Val Ile Ser Leu Gly Leu Phe Leu Thr Leu 245 250 255 Leu Thr Leu ThrIle Asp Ala Ser Lys His Arg Met Ile Tyr Leu Leu 260 265 270 Ser Val GlnThr Met Gly Val Phe Ile Ile His Thr Tyr Val Met Gln 275 280 285 Ile TrpGln Lys Leu Ile Gly Phe Asn Ile Val Gly Ala His Leu Phe 290 295 300 PhePro Val Phe Thr Leu Val Ile Ser Phe Leu Ile Ser Met Ile Leu 305 310 315320 Met Lys Ile Pro Tyr Ile Asn Arg Ile Val Lys Leu 325 330 34 366 PRTStreptococcus macedonicus 34 Met Tyr Asp Tyr Leu Ile Val Gly Ala Gly LeuSer Gly Ala Ile Phe 1 5 10 15 Ala Gln Glu Ala Thr Lys Arg Gly Lys LysVal Lys Val Ile Asp Lys 20 25 30 Arg Asp His Ile Gly Gly Asn Ile Tyr CysGlu Asp Val Glu Gly Ile 35 40 45 Asn Val His Lys Tyr Gly Ala His Ile PheHis Thr Ser Asn Lys Lys 50 55 60 Val Trp Asp Tyr Val Asn Gln Phe Ala GluPhe Asn Asn Tyr Ile Asn 65 70 75 80 Ser Pro Ile Ala Asn Tyr Lys Gly SerLeu Tyr Asn Leu Pro Phe Asn 85 90 95 Met Asn Thr Phe Tyr Ala Met Trp GlyThr Lys Thr Pro Gln Glu Val 100 105 110 Lys Asp Lys Ile Ala Glu Gln ThrAla Asp Met Lys Asp Val Glu Pro 115 120 125 Lys Asn Leu Glu Glu Gln AlaIle Lys Leu Ile Gly Pro Asp Ile Tyr 130 135 140 Glu Lys Leu Ile Lys GlyTyr Thr Glu Lys Gln Trp Gly Arg Ser Ala 145 150 155 160 Thr Asp Leu ProPro Phe Ile Ile Lys Arg Leu Pro Val Arg Leu Thr 165 170 175 Phe Asp AsnAsn Tyr Phe Asn Asp Arg Tyr Gln Gly Ile Pro Ile Gly 180 185 190 Gly TyrAsn Val Ile Ile Glu Asn Met Leu Gly Asp Val Glu Val Glu 195 200 205 LeuGly Val Asp Phe Phe Ala Asn Arg Glu Glu Leu Glu Ala Ser Ala 210 215 220Glu Lys Val Val Phe Thr Gly Met Ile Asp Gln Tyr Phe Asp Tyr Lys 225 230235 240 His Gly Glu Leu Glu Tyr Arg Ser Leu Arg Phe Glu His Glu Val Leu245 250 255 Asp Glu Glu Asn His Gln Gly Asn Ala Val Val Asn Tyr Thr GluArg 260 265 270 Glu Ile Pro Tyr Thr Arg Ile Ile Glu His Lys His Phe GluTyr Gly 275 280 285 Thr Gln Pro Lys Thr Val Ile Thr Arg Glu Tyr Pro AlaAsp Trp Lys 290 295 300 Arg Gly Asp Glu Pro Tyr Tyr Pro Ile Asn Asp GluLys Asn Asn Ala 305 310 315 320 Met Phe Ala Lys Tyr Gln Glu Glu Ala GluLys Asn Asp Lys Val Ile 325 330 335 Phe Cys Gly Arg Leu Ala Asp Tyr LysTyr Tyr Asp Met His Val Val 340 345 350 Ile Glu Arg Ala Leu Glu Val ValGlu Lys Glu Phe Thr Ile 355 360 365 35 224 PRT Streptococcus macedonicus35 Met Ile Glu Lys Asn Ala Pro Trp Val Asn Asn Val Tyr Leu Ile Thr 1 510 15 Asn Gly Gln Lys Pro Asp Trp Leu Asn Leu Glu His Pro Lys Leu Lys 2025 30 Leu Val Thr His Arg Glu Phe Met Pro Lys Glu Tyr Leu Pro Thr Tyr 3540 45 Asn Ser Ala Ala Ile Glu Leu Asn Leu His His Ile Glu Gly Leu Ser 5055 60 Glu Asn Tyr Leu Tyr Phe Asn Asp Asp Thr Tyr Leu Ile Arg Asp Ser 6570 75 80 Gln Pro Ser Asp Phe Tyr Lys Asn Gly Gln Pro Lys Leu Leu Ala Val85 90 95 Tyr Asp Ala Leu Val Pro Trp Pro Pro Phe Thr Asn Thr Tyr His Asn100 105 110 Asn Val Glu Leu Ile Tyr Arg His Phe Pro Asn Lys Lys Ala LeuLys 115 120 125 Ser Ser Pro Trp Lys Phe Phe Asn Phe Arg Tyr Gly Ser LeuVal Leu 130 135 140 Lys Asn Leu Leu Leu Leu Pro Trp Gly Pro Thr Arg TyrVal Asn Gln 145 150 155 160 His Leu Pro Val Pro Met Lys Lys Ser Thr LeuAla His Leu Trp Glu 165 170 175 Ile Glu Gly Glu Thr Leu Asp Lys Thr SerArg Asn Pro Ile Arg Asp 180 185 190 Tyr Gly Val Asp Val Asn Gln Tyr IleCys Gln His Trp Gln Ile Glu 195 200 205 Ser Asn Gln Phe Tyr Pro Met SerLys Ser Phe Gly Glu Thr Ile Gly 210 215 220 36 391 PRT Streptococcusmacedonicus 36 Met Thr Gln Phe Thr Thr Glu Leu Leu Asn Phe Leu Ala GlnLys Gln 1 5 10 15 Asp Ile Asp Glu Phe Phe Arg Thr Ser Leu Glu Thr AlaMet Asn Asp 20 25 30 Leu Leu Gln Ala Glu Leu Ser Ala Phe Leu Gly Tyr GluPro Tyr Asp 35 40 45 Lys Leu Gly Tyr Asn Ser Gly Asn Ser Arg Asn Gly SerTyr Ala Arg 50 55 60 Lys Phe Glu Thr Lys Tyr Gly Thr Val Gln Leu Ser IlePro Arg Asp 65 70 75 80 Arg Asn Gly Asn Phe Ser Pro Ala Leu Leu Pro AlaTyr Gly Arg Arg 85 90 95 Asp Asp His Leu Glu Glu Met Val Ile Lys Leu TyrGln Thr Gly Val 100 105 110 Thr Thr Arg Glu Ile Ser Asp Ile Ile Glu ArgMet Tyr Gly His His 115 120 125 Tyr Ser Pro Ala Thr Ile Ser Asn Ile SerLys Ala Thr Gln Glu Asn 130 135 140 Val Ala Thr Phe His Glu Arg Ser LeuGlu Ala Asn Tyr Ser Val Leu 145 150 155 160 Phe Leu Asp Gly Thr Tyr LeuPro Leu Arg Arg Gly Thr Val Ser Lys 165 170 175 Glu Cys Ile His Ile AlaLeu Gly Ile Thr Pro Glu Gly Gln Lys Ala 180 185 190 Val Leu Gly Tyr GluIle Ala Pro Asn Glu Asn Asn Ala Ser Trp Ser 195 200 205 Thr Leu Leu AspLys Leu Gln Asn Gln Gly Ile Gln Gln Val Ser Leu 210 215 220 Val Val ThrAsp Gly Phe Lys Gly Leu Glu Glu Ile Ile Asn Gln Ala 225 230 235 240 TyrPro Leu Ala Lys Gln Gln Arg Cys Leu Ile His Ile Ser Arg Asn 245 250 255Leu Ala Ser Lys Val Lys Arg Ala Asp Arg Ala Val Ile Leu Glu Gln 260 265270 Phe Lys Thr Ile Tyr Arg Ala Glu Asn Leu Glu Met Ala Val Gln Ala 275280 285 Leu Glu Asn Phe Ile Ser Glu Trp Lys Pro Lys Tyr Arg Lys Val Met290 295 300 Glu Ser Leu Glu Asn Thr Asp Asn Leu Leu Thr Phe Tyr Gln PhePro 305 310 315 320 Tyr Gln Ile Trp His Ser Ile Tyr Ser Thr Asn Leu IleGlu Ser Leu 325 330 335 Asn Lys Glu Ile Lys Arg Gln Thr Lys Lys Lys IleLeu Phe Pro Asn 340 345 350 Glu Glu Ala Leu Gly Arg Tyr Leu Val Thr LeuPhe Glu Asp Tyr Asn 355 360 365 Phe Lys Gln Ser Gln Arg Thr His Lys GlyPhe Gly Gln Cys Ala Asp 370 375 380 Thr Leu Glu Ser Leu Phe Asp 385 39037 300 DNA Streptococcus macedonicus 37 atgggaattg aaatttttat tcgtaacccaaaaggcatta ccttgactaa ggatggcgtt 60 aggtttcttt cttatgcgcg ccaaattttagaacaaacag ctcttttaga ggaacgttat 120 aagagcaaaa atacaaaccg agaactgtttagcgtatctt cacagcacta tgctttcgtt 180 gtcaatgctt ttgtttcgct tttagaaggaacagatatgt cacgttatga gcttttcctt 240 cgcgaaacac gaacatatga aattattgatgatgttaaga atttccgttc agaaattggc 300

What is claimed is:
 1. A biologically pure culture of a lactic acidbacteria strain that comprises a 16S ribosomal RNA comprising anucleotide sequence that is SEQ ID NO:1 or a homologue thereof having1-8 nucleotide substitutions, deletions, or additions, and comprisingcocci morphology, a growth optimum in the range of about 28° C. to about45° C., and the ability to ferment D-galactose, D-glucose, D-fructose,D-mannose, and N-acetyl(D)-glucosamine, salicin, cellobiose, maltose,lactose, sucrose and raffinose, and imparts a viscosity of greater than100 mPa.s at a shear rate of about 293 s⁻¹ when used to fermentsemi-skimmed milk at 38° C. at up to a pH 5.2.
 2. The strain of claim 1,wherein the 16S ribosomal RNA is SEQ ID NO:1.
 3. The strain of claim 1,wherein the strain produces an exopolysaccharide comprising a chain ofglucose, galactose and N-acetylglucosamine in a proportion of 3:2:1respectively.
 4. The strain of claim 1, wherein the total proteinprofile obtained after culture of the bacterium in an MRS medium for 24h at 28° C., extraction of the total proteins and migration of theproteins on an SDS-PAGE electrophoresis gel, exhibits a degree ofPearson correlation of at least 78 with respect to the profile obtainedunder identical conditions with the strain of lactic acid bacterium CNCMI-1920 or I-1
 926. 5. The strain of claim 1, further comprising anucleotide sequence that encodes the polypeptides identified by SEQ IDNOS:18, 20, 22-24, 27, 28, 32, and 34 (SM-epsA, C, E-G, J, K, 0, and Q),wherein the strain produces an exopolysaccharide comprising a chain ofglucose, galactose and N-acetylglucosamine in a proportion of 3:2:1respectively.
 6. A dietary or pharmaceutical composition comprising apolysaccharide secreted by the strain of claim
 1. 7. The composition ofclaim 6, wherein the polysaccharide secreted has a chain of glucose,galactose and N-acetylglucosamine in a proportion of 3:2:1 respectively.8. The composition of claim 6, wherein the polysaccharide is hydrolyzedand comprises polysaccharides that have predominantly 3 to 10 sugarunits.
 9. The composition of claim 7, which is a hypoallergenic infantcomposition.
 10. A dietary or pharmaceutical comprising a strain oflactic acid bacterium according to claim
 1. 11. A method of preparing adietary or pharmaceutical composition comprising: adding a lactic acidbacterium strain according to claim 1 to a dairy product to prepare thecomposition.
 12. The method of claim 11, wherein the dairy productcomprises milk.
 13. A biologically pure culture of a lactic acidbacteria strain, wherein the bacteria strain comprises nucleotidesequences which encode polypeptides identified by SEQ ID NOS:18, 20,22-24, 27, 28, 32, and 34 (SM-epsA, C, E-G, J, K, O, and Q), and thestrain produces an exopolysaccharide comprising a chain of glucose,galactose and N-acetylglucosamine in a proportion of 3:2:1 respectively.14. The strain of claim 13, having a total protein profile, wherein thetotal protein profile obtained after culture of the bacterium in an MRSmedium for 24 h at 28° C., extraction of the total proteins andmigration of the proteins on an SDS-PAGE electrophoresis gel, exhibits adegree of Pearson correlation of at least 78 with respect to the profileobtained under identical conditions with the strain of lactic acidbacterium CNCM I-1920 or I-1026.
 15. The strain of claim 13, wherein thestrain further comprises a nucleotide sequence which encodes thepolypeptides identified by SEQ ID NOS:21, 25-26, and 33 (SM-epsD, H-I,and P).
 16. The strain of claim 15, wherein the strain further comprisesa nucleotide sequence which encodes the polypeptides identified by SEQID NOS:19 and 29-31 (SM-epsB and L-N).
 17. The strain of claim 16,wherein the strain comprises SEQ ID NO:4.
 18. A dietary orpharmaceutical composition comprising a polysaccharide secreted by thestrain of claim
 13. 19. A method of preparing a dietary orpharmaceutical composition comprising: adding a lactic acid bacteriumstrain according to claim 13 to a dairy product to prepare thecomposition.
 20. The method of claim 19 wherein the dairy productcomprises milk.
 21. An isolated nucleotide sequence that encodes apeptide identified by SEQ ID NOS:18, 20, 22- 27, 28, 32, 34, or 35(SM-epsA, C, E-K, O, Q, or R).
 22. A transformed microorganismcomprising a nucleotide sequence of claim 21, wherein the microorganismproduces an exopolysaccharide comprising a chain of glucose, galactoseand N-acetylglucosamine in a proportion of 3:2:1 respectively.